Novel peptide agonists of GLP-1 activity

ABSTRACT

Novel peptide agonists of GLP-1 activity useful for lowering blood glucose levels. The novel peptides comprise variants of the GLP-1 or the exendin-4 polypeptide sequence and are pharmacologically active and stable. These peptides are useful in the treatment of diseases that benefit from regulation of excess levels of blood glucose and/or regulation of gastric emptying, such as diabetes and eating disorders.

FIELD OF THE INVENTION

[0001] The present invention relates to novel peptide agonists of GLP-1activity. More specifically the invention relates to novel peptides thatlower blood glucose levels comprising variants of the exendin-4polypeptide sequence and peptide conjugates comprising variants of theGLP-1 or the exendin-4 polypeptide sequences which are pharmacologicallyactive and stable, and as agonists of GLP-1 activity are useful in thetreament of diseases that benefit from regulation of excess levels ofblood glucose and/or regulation of gastric emptying, such as diabetesand eating disorders. The present invention also relates to methods ofpreparing said novel peptides, a composition, e.g., a pharmaceuticalcomposition, comprising a peptide of the invention and a physiologicallyacceptable carrier, to said peptide for use in therapy, a method oftreating a disorder and to the use of said peptide for the manufactureof a pharmaceutical composition for use in therapy.

BACKGROUND OF THE INVENTION

[0002] A number of hormones that lower blood glucose levels are releasedfrom the gastrointestinal mucosa in response to the presence andabsorption of nutrients in the gut. These include gastrin, secretin,glucose-dependent insulinotropic polypeptide (GIP) and glucagon-likepeptide-1 (GLP-1). The most potent substance known is GLP-1 (Ørskov,1992, Diabetologia 35:701-711). Glucagon-like peptide 1 (GLP-1) is aproduct of proglucagon, a 180 amino acid peptide (Drucker, 1998,Diabetes 47:159-169). The overall sequence of proglucagon contains the29-amino acid sequence of glucagon, the 36 or 37 amino acid sequence ofGLP-1 and the 34 amino acid sequence of glucagon-like peptide-2 (GLP-2),an intestinotrophic peptide. GLP-1 has a number of functions. It is aphysiological hormone that enhances the effect on insulin secretion innormal humans and is therefore an incretin hormone. In addition, GLP-1also lowers glucagon concentrations, slows gastric emptying, stimulates(pro)insulin biosynthesis, and enhances insulin sensitivity (Nauck,1997, Horm. Metab. Res. 47:1253-1258). The peptide also enhances theability for the β-cells to sense and respond to glucose in subjects withimpaired glucose tolerance (Byrne, 1998, Eur. J. Clin. Invest.28:72-78). The insulinotropic effect of GLP-1 in humans increases therate of glucose disappearance partly because of increased insulin levelsand partly because of enhanced insulin sensitivity (D'Alessio, 1994,Eur. J. Clin. Invest. 28:72-78). This has placed GLP-1 as a promisingagent for treatment for type II diabetes. Active fragments of GLP-1 havebeen found to be GLP-1(7-36) (SEQ ID NO: 114) and GLP-1(7-37) (SEQ IDNO: 124).

[0003] However, a major pharmacological problem with native GLP-1 is itsshort half-life. In humans and rats, GLP-1 is rapidly degraded bydipeptidyl peptidase-IV (DPP-IV) into GLP-1(9-36)amide (SEQ ID NO: 125),acting as an endogenous GLP-1 receptor antagonist (Deacon, 1998,Diabetologia 41:271-278). Several strategies circumventing this problemhave been proposed, some using inhibitors of DPP-IV and others DPP-IVresistant analogues of GLP-1 (7-36)amide (SEQ ID NO: 114) (Deacon, 1998,Diabetologia 41:271-278; Deacon et al., 1998, Diabetes 47:764-769;Ritzel, 1998, J. Endocrinol. 159:93-102; U.S. Pat. No. 5,545,618;Pederson, 1998, Diabetes 47:1253-1258).

[0004] Exendins, another group of peptides that lower blood glucoselevels have some sequence similarity (53%) to GLP-1[7-36]NH₂ (SEQ ID NO:114) (Goke et al., 1993, J. Biol. Chem. 268:19650-55). The exendins arefound in the venom of Helodermatidae or beaded lizards (Raufman, 1996,Reg. Peptides 61:1-18). Exendin-3 is present in the venom of Helodermahorridum, the Mexican beaded lizard and exendin-4 is present in thevenom of Heloderma suspectum, the Gila monster. Exendin-4 differs fromexendin-3 at just positions two and three. The cDNA encoding theexendin-4 precursor protein, a 47 amino acid peptide fused to the aminoterminus of exendin-4 has been cloned and sequenced (Pohl et al., 1998,J. Biol. Chem. 273:9778-9784 and WO98/35033). Both exendin-3 andexendin-4 stimulate an increase in cellular cAMP production in guineapig pancreatic acinar cells by interacting with exendin receptors(Raufman, 1996, Reg. Peptides 61:1-18). Exendin-3 causes a biphasicincrease in cellular cAMP production, but a monophasic increase inamylase release in pancreatic acinar cells. In contrast, exendin-4causes a monophasic increase in cAMP production and does not alteramylase release.

[0005] Exendin-4 is a strong GLP-1 receptor agonist on isolated ratinsulinoma cells (Goke et al., 1993, J. Biol. Chem. 268:19650-55). Thisis expected as the (His Ala) domain of GLP-1 recognised by DPP-IV is notpresent in exendin-4 (Goke et al., 1993, J. Biol. Chem. 268:19650-55).Binding of [¹²⁵I]GLP-1 to the nucleus of the solitary tract wasinhibited concentration-dependently by unlabelled GLP-1 and[Tyr39]exendin-4 (SEQ ID NO: 126) with Ki values of 3.5 and 9.4 nMrespectively, and similar values are found in cell lines (Goke et al.,1995, Eur. J. Neurosci. 7:2294-2300 and Goke et al., 1993, J. Biol.Chem. 268:19650-55). Further, exendin-4 given systemically lowers bloodglucose levels by 40% in diabetic db/db mice (WO99/07404). Recently,Grieg et al. (1999, Diabetologia 42:45-50) has shown a long lastingblood glucose lowering effect of once daily intraperitoneal injection ofexendin-4 to diabetic ob/ob mice). U.S. Pat. No. 5,424,286 disclosesthat a considerable portion of the N-terminal sequence is essential inorder to preserve insulinotropic activity (exendin-4(1-31) (SEQ ID NO:127) and Y³¹-exendin-4(1-31)) (SEQ ID NO: 148) whereas an N-terminallytruncated exendin (exendin-4(9-39) (SEQ ID NO: 128) has inhibitoryproperties.

[0006] The use of exendin-3, exendin-4 and exendin agonists has beenproposed for the treatment of diabetes mellitus, reducing gastricmotility and delaying gastric emptying and the prevention ofhyperglycemia (U.S. Pat. No. 5,424,286, WO98/05351) as well as for thereduction of food intake (WO98/30231). There has been proposed ways ofobtaining novel compounds by modifying the native exendin sequences. Oneway is to attach lipophilic substituents to the molecule, e.g. asdescribed in WO 99/43708 which discloses derivatives of exendin withjust one lipophilic substituent attached to the C-terminal amino acidresidue.

[0007] A major approach has been to devise exendin analoguescharacterised by amino acid substitutions and/or C-terminal truncationof the native exendin-4 sequence. This approach is represented by thecompounds of WO99/07404, WO 99/25727 and WO 99/25728. WO99/07404discloses exendin agonists having a general formula I that defines apeptide sequence of 39 amino acid residues with Gly Thr in positions4-5, Ser Lys Gin in positions 11-13, Glu Gin Glu Ala Val Arg Leu inpositions 15-21, Leu Lys Asn Gly Gly in positions 26-30, Ser Ser Gly Alain positions 32-35, and wherein the remaining positions may be occupiedby wild-type exendin amino acid residues or may be occupied by specifiedamino acid substitutions. The formula I does not cover any exendinagonists or analogues having specific amino acid deletions and/or beingconjugates as described herein, such as the novel compoundsdesPro³⁶-exendin-4(1-39) (SEQ ID NO: 101), exendin-4(1-39)-K₆ (SEQ IDNO: 92) or desPro³⁶-exendin-4(1-39)-K₆(SEQ ID NO: 93).

[0008] WO 99/25727 discloses exendin agonists having a general formula Ithat defines a peptide sequence of from 28 to 38 amino acid residueswith Gly in position 4 and Ala in position 18, and wherein the remainingpositions may be occupied by wild-type exendin amino acid residues ormay be occupied by specified amino acid substitutions. Formula I doesnot comprise a peptide sequence having Ser as the C-terminal amino acidand exendin agonists or analogues having specific amino acid deletionsand/or being conjugates as described herein, such as the novel compoundsdesPro³⁶-exendin-4(1-39) (SEQ ID NO: 101), exendin-4(1-39)-K₆ (SEQ IDNO: 92) or desPro³⁶-exendin-4(1-39)-K₆(SEQ ID NO: 93). Further, formulaII of WO 99/25727 defines a peptide sequence similar to formula I, butincluding exendin derivatives having a C(1-10)alkanoyl orcycloalkylalkanoyl substituent on lysine in position 27 or 28.

[0009] When treating inappropriate post-prandial blood glucose levelsthe compounds are administered frequently, for example one, two or threetimes a day.

[0010] WO 99/25728 discloses exendin agonists having a general formula Ithat defines a peptide sequence of from 28 to 39 amino acid residueswith fixed Ala in position 18, and wherein the remaining positions maybe occupied by wild-type exendin amino acid residues or may be occupiedby specified amino acid substitutions. Said exendin agonists allcorrespond to a truncated exendin analogue having a varying degree ofamino acid substitutions. Peptide sequences of from 34 to 38 amino acidresidues do not have Ser C-terminally. A peptide sequence of 39 aminoacid residues may have either Ser or Tyr C-terminally, but no furtherresidues. Exendin agonists or analogues having specific amino aciddeletions and/or being conjugates according to the invention describedherein are not comprised by formula I. Further, formula II defines apeptide sequence similar to formula I, but including exendin derivativeshaving a C(1-10)alkanoyl or cycloalkylalkanoyl substituent on lysine inposition 27 or 28.

[0011] WO 99/46283 (published 16.09.99) discloses peptide conjugatescomprising a pharmacologically active peptide X and a stabilisingpeptide sequence Z of 4-20 amino acid residues covalently bound to X,where said conjugates are characterised in having an increased half-lifecompared to the half-life of X. X may be exendin-4 or exendin-3.

OBJECTIVE OF THE INVENTION

[0012] There is a need for compounds that lower blood glucose levels inmammals, and are stable and effective. Therefore, it is an objective ofthe invention to provide novel compounds that lower blood glucose levelsin mammals. Ideally, these should be effective when administered orally.It is a further object of the invention to provide novel peptideagonists of GLP-1 activity and/or exendin-4 activity. It is a stillfurther purpose of the invention to provide peptide agonists of GLP-1activity and/or exendin-4 activity having an increased half-life and/ora decreased clearance.

SUMMARY OF THE INVENTION

[0013] The invention is directed to a peptide conjugate comprising apeptide X selected from the group consisting of

[0014] (a) an exendin having at least 90% homology to exendin-4;

[0015] (b) a variant of said exendin wherein said variant comprises amodification selected from the group consisting of between one and fivedeletions at positions 34-39 and contains a Lys at position 40 having alipophilic substituent; or

[0016] (c) GLP-1 (7-36) (SEQ ID NO: 114) or GLP-1 (7-37) (SEQ ID NO:124) having at least one modification selected from the group consistingof:

[0017] (i) substitution of D-alanine, glycine or alpha-amino isobutyricacid for alanine at position 8 and

[0018] (ii) a lipophilic substituent,

[0019]  and Z, a peptide sequence of 4-20 amino acid units covalentlybound to said variant, wherein each amino acid unit in said peptidesequence, Z is selected from the group consisting of Ala, Leu, Ser, Thr,Tyr, Asn, Gin, Asp, Glu, Lys, Arg, His, Met, Orn, and amino acid unitsof the general formula I

—NH—C(R¹)(R²)—C(═O)—  (I)

[0020]  wherein R¹ and R² are selected from the group consisting ofhydrogen, C₁₋₆-alkyl, phenyl, and phenyl-methyl, wherein C₁₋₆-alkyl isoptionally substituted with from one to three substituents selected fromhalogen, hydroxy, amino, cyano, nitro, sulfono, and carboxy, and phenyland phenyl-methyl is optionally substituted with from one to threesubstituents selected from C₁₋₆-alkyl, C₂₋₆-alkenyl, halogen, hydroxy,amino, cyano, nitro, sulfono, and carboxy, or R¹ and R² together withthe carbon atom to which they are bound form a cyclopentyl, cyclohexyl,or cycloheptyl ring, e.g. 2,4-diaminobutanoic acid and2,3-diaminopropanoic acid, with the proviso that X is not exendin-4 orexendin-3.

[0021] The peptide X is further characterised in being effective inimproving glucose tolerance in a diabetic mammal.

[0022] Furthermore, the invention is directed to a novel variant of aparent exendin, wherein said parent exendin has an amino acid sequencehaving at least an 90% homology to exendin-4 and wherein said variantlowers the blood glucose level in a mammal, binds to a GLP-1 receptorand has at least one modification selected from the group consisting of(a) between one and five deletions at positions 34-38, and (b) containsa Lys at position 40 having a lipophilic substituent attached to theepsilon amino group of said lysine.

BRIEF DESCRIPTION OF THE FIGURES

[0023]FIG. 1 shows the effect of Compound 1 (SEQ ID NO:101) (desPro³⁶-exendin-4(1-39)-NH₂) on blood glucose levels of mice, cf. Example25.

[0024]FIG. 2 shows the effect of Compound 2 (SEQ ID NO:93) (desPro³⁶-exendin-4(1-39)-Lys₆-NH₂ on the blood glucose levels of mice, cf.Example 25.

[0025]FIG. 3 shows the effect of Compound 5 (SEQ ID NO:89) (Gly⁸,Lys³⁷(palmitoyl)-GLP1-(7-36)(Human)-(Lys)₇-NH₂ on the blood glucoselevels of mice, cf. Example 25.

[0026]FIG. 4 shows in vivo degradation kinetics in rabbits after i.v.injection of 1 μmol/kg of Compound 4 and Compound (iii), cf. Example 27.

[0027]FIG. 5 is a plot of AUC (area under the curve) values (mean±SEM)for Compounds 2, 14-16, 18 and 19 in an oral glucose tolerance test(OGTT), cf. Example 28.

[0028]FIG. 6 shows a synthetic cDNA constructed for heterolog expressionof Compound 2 in yeast. The new construct was designated pYES0010, cf.Example 20.

[0029]FIG. 7 is a plot of dose-response on GTT in db/db mice based onrelative AUC_(0-240 min) values (mean±SEM) for Compound 2 and Compound(i), cf. Example 29.

[0030]FIG. 8 shows the effects of a maximal dose of Compound 2, i.e. 100nmol/kg i.p., on the oral glucose tolerance test (OGTT) whenadministered up to 24 hours before the OGTT.

DETAILED DESCRIPTION OF THE INVENTION

[0031] The compounds of the present invention include hitherto unknowndeletion variants of a parent exendin. In contrast to known substitutionand/or truncation variants of exendin-4(1-39) the novel compounds arebelieved to exhibit a stabilised alpha-helix structure with superiorstability properties and unreduced or enhanced binding properties.Moreover, conjugation of the novel variants, modifiedGLP-1(7-36)-NH₂(SEQ ID NO: 114), and modified GLP-1(7-37) (SEQ ID NO:124) to specific short peptide sequences (Z) render stability to thesecompounds without compromising the pharmacological properties. Theseconjugations confer in vivo stability and hydrophilicity to the peptidemolecule. The Z is composed of amino-acid residues, and has alone nostructural characteristics in terms of α-helix conformation. However,from studies using both circular dichroism and nuclear magneticresonance (NMR) spectroscopy, addition of Z dramatically alters thestructural characteristics of some peptides as evidenced by theincreased amount of α-helix conformation in the peptide. For example,circular dichroism demonstrated that a Z-modified (Gly⁸)-GLP-1 (SEQ IDNO: 87) had much more α-helix conformation than (Gly⁸)-GLP-1(SEQ ID NO:87). Together with the pharmacological results, the structural analysessuggest that Z is modifying the conformation of the peptide leading tohigher enzyme-stability, but without losing its potency. Also thephysical and chemical properties of peptides may be altered considerablyby Z-modification with resulting impact on pharmacological formulationstrategy.

[0032] Exendin Variants

[0033] The exendin variant of the present invention is a variant of aparent exendin peptide having at least about 90% homology and mostpreferably at least about 95% to exendin-4, which have exendin activity,e.g., lowers the blood glucose level in a mammal and binds to a GLP-1receptor. In a preferred embodiment, the parent exendin peptide has anamino acid sequence which differs by five amino acids, preferably byfour amino acids, more preferably by three amino acids, even morepreferably by two amino acids, and still more preferably by one aminoacid residue from the amino acid sequence of exendin-4(1-39) (SEQ ID NO:102).

[0034] In one embodiment, the exendin variant comprises between one andfive deletions at positions 34-38. Preferably the variant comprisesbetween 1 and 4 deletions at positions 34-38, more preferably between 1and 3 deletions at positions 36-38. Preferably the parent exendin isexendin-4, and a preferred variant included as peptide X in the peptideconjugates herein has an amino acid sequence wherein 1, 2 or 3 of thePro residues in positions 36, 37 and 38 have been deleted from the aminoacid sequence of exendin-4 and preferably from the amino acid sequenceof exendin-4(1-39) (SEQ ID NO: 102).

[0035] Coupling of a Z sequence to the X peptide herein is believed toincrease the stability of these compounds. Proline is a rigid amino acidthat may interfere with the effect of Z to stabilise the structure ofthe X peptide. Deletion of one, two or all of the proline amino acids inpositions 36, 37 and 38 of the exendin backbone is therefore preferredin the peptide conjugates comprising a variant of a parent exendinaccording to the invention, as long as the efficacy of said conjugatesas measured in, e.g. an oral glucose tolerance test (OGTT) in diabeticdb/db mice, is not negatively affected.

[0036] In another embodiment, the variant comprises an additionalresidue at position 40, a lysine residue which comprises a lipophilicsubstituent bound to the epsilon amino group of lysine via an amidebond. The lipophilic substituent may be the acyl group of astraight-chain or branched fatty acid or a straight-chain or branchedalkane α,ω-dicarboxylic acid. The acyl group may have the formulaCH₃(CH₂)_(n)CO—, wherein n is an integer from 4-38 and preferably from4-24. In a specific embodiment, the acyl group is selected from thegroup consisting of CH₃(CH₂)₆CO—, CH₃(CH₂)₈CO—, CH₃(CH₂)₁₀CO—,CH₃(CH₂)₁₂CO—, CH₃(CH₂)₁₄CO—, CH₃(CH₂)₁₆CO—, CH₃(CH₂)₁₈CO—,CH₃(CH₂)₂₀CO—, and CH₃(CH₂)₂₂CO—. The acyl group may have the formulaHOOC(CH₂)_(m)CO—, wherein n is an integer from 4-38 and preferably from4-24. In a specific embodiment, the acyl group is selected from thegroup consisting of HOOC(CH₂)₁₄CO—, HOOC(CH₂)₁₆CO—, HOOC(CH₂)₁₈CO—,HOOC(CH₂)₂₀CO— and HOOC(CH₂)₂₂CO—. In a more specific embodiment, thelipophilic substituent is selected from the group consisting oftetradecanoyl, ω-carboxynonadecanoyl, 7-deoxycholoyl, choloyl, palmitoyland lithocholyl. In a most specific embodiment, the lipophilicsubstituent is palmitoyl.

[0037] Alternatively, the liphophilic substituent may have an NH group.Specific embodiments include but are not limited to the formulaeCH₃(CH₂)_(a)((CH₂)_(b)COOH)CHNHCO(CH₂)₂CO— wherein a and b are integersand a+b is an integer of from 8 to 33, preferably from 12 to 28;CH₃(CH₂)_(n)CONHCH(COOH) (CH₂)₂CO— wherein c is an integer of from 10 to24; CH₃(CH₂)_(d) CONHCH(CH₂)₂ (COOH)CO— wherein d is an integer of from8 to 24; COOH(CH₂)_(e)CO— wherein e is an integer of from 8 to 24;—NHCH(COOH)(CH₂)₄NHCO(CH₂)_(f)CH₃ wherein f is an integer of from 8 to18; —NHCH(COOH)(CH₂)₄NHCOCH(CH₂)₂COOH)NHCO(CH₂)_(g)CH₃ wherein g is aninteger of from 10 to 16; and —NHCH(COOH)(CH₂)₄NHCO(CH₂)₂CH(COOH)NHCO(CH2)_(h)CH₃ wherein h is an integer of 0 or from 1 to 22 and preferablyfrom 10 to 16.

[0038] The exendin variants having a lysine residue at position 40carrying a lipophilic substituent optionally further comprise betweenone and five deletions, preferably between one and three deletions, atpositions 34 to 39, preferably at positions 34-38, such as [des Ser³⁹,Lys⁴⁰ (palmitoyl)]exendin-4(1-39) (SEQ ID NO: 107), [des Pro³⁶,Lys⁴⁰(palmitoyl)]exendin-4(1-39) (SEQ ID NO: 110) and [des Pro³⁶,Lys⁴⁰(palmitoyl)]exendin-4(1-40) (SEQ ID NO: 152).

[0039] The variant may be in a most specific embodiment selected fromthe group consisting of:

[0040] Compound 1: des Pro³⁶-exendin-4(1-39)-NH₂ (SEQ ID NO: 101),

[0041] des Pro³⁶-exendin-4(1-40)-NH₂ (SEQ ID NO: 139),

[0042] Compound 14: des Pro³⁶, Pro³⁷,Pro³⁸-exendin (1-39)-NH₂(SEQ ID NO:132),

[0043] des Pro³⁶, Pro³⁷,Pro³⁸-exendin 4(40)-NH₂(SEQ ID NO: 140),

[0044] des Pro³⁶, Pro³⁷-exendin-4(1-39)-NH₂(SEQ ID NO: 130),

[0045] des Ala³⁵-exendin-4(1-39)-NH₂ (SEQ ID NO:105),

[0046] des Gly 34-exendin-4(1-39)-NH₂ (SEQ ID NO: 106),

[0047] des Ser³⁹-(Lys⁴⁰ (palmitoyl))exendin-4(1-39)-NH₂ (SEQ ID NO:107),

[0048] des Gly³⁴-(Lys⁴⁰(palmitoyl))exendin-4(1-39)-NH₂ (SEQ ID NO:108),

[0049] des Ala³⁵(Lys⁴⁰ (palmitoyl))exendin-4(1-39)-NH₂ (SEQ ID NO:109),

[0050] des Pro³⁶-(Lys⁴⁰ (palmitoyl))exendin-4(1-39)-NH₂ (SEQ ID NO:110),and the free acid thereof and a pharmaceutically acceptable saltthereof.

[0051] Modified GLP-1

[0052] A preferred modified GLP-1 included as peptide X in the peptideconjugates herein has an amino acid sequence of GLP-1 (7-36)-NH₂ (SEQ IDNO: 114) or GLP-1 (7-37) (SEQ ID NO: 124) having a substitution ofglycine for alanine at position 8. Alternatively, a preferred modifiedGLP-1 has an amino acid sequence of GLP-1 (7-36) (SEQ ID NO: 114) orGLP-1 (7-37) (SEQ ID NO: 124) having a substitution of glycine foralanine at position 8 and a lipophilic substituent, preferablypalmitoyl, on one lysine residue at position 26, 34 or 37. Thelipophilic substituent is preferably attached to the epsilon amino groupof said lysine and includes the specific embodiments described above forthe exendin variants. The modified GLP-1(7-36) (SEQ ID NO: 114) orGLP-1(7-37) (SEQ ID NO: 124) used as X in the conjugates of theinvention may be those cited in WO 99/43707 and WO 98/08871 comprising alipophilic substituent or, more preferably those GLP-1 analogues havinga glycine substitution at position 8. Preferred peptides X are

[0053] Gly⁸-GLP-1(7-36) (SEQ ID NO: 87),

[0054] Gly⁸-GLP-1(7-37) (SEQ ID NO:123), and

[0055] Gly⁸-GLP-1(7-36)-Lys³⁷(palmitoyl) (SEQ ID NO: 147).

[0056] The compounds of the invention having a lipophilic substituentwould have a more protracted profile of action than the parent peptidesas demonstrated for GLP-1 derivatives in WO 98/08871.

[0057] Peptide Conjugates

[0058] The peptide sequence Z may be bound to the C-terminal or theN-terminal of the peptide sequence, X, or two peptide sequences may bebound individually to both the C- and N-terminal of X. In case thenative peptide X possesses a free C-terminal carboxylic acid, thepeptide sequence Z may be attached to either the C-terminal of thepeptide X or to the N-terminal of the peptide X, or the C- andN-terminal of X may both be bound to each individual peptide sequence Z.Alternatively, Z may be bound to the nitrogen atom on the side chain oflysine, histidine or arginine or a carbonyl function on the side chainof glutamic acid or aspartic acid anywhere within the peptide sequenceX. In one embodiment, Z may be attached to X within the sequence and tothe N- and/or C-terminal of X. Whether the sequence should be attachedto the peptide sequence X at its C-terminal, at its N-terminal, or both,or within the peptide sequence X depends on the specific peptide X andcan be easily determined by the person skilled in the art. Preferably, Xis bound to Z via a peptide bond and preferably at the C-terminal of X.

[0059] One aspect of the invention is directed to a peptide conjugatecomprising a peptide X which reduces the blood glucose level in amammal, wherein X is (a) an exendin having at least 90% homology toexendin-4; (b) a variant of said exendin wherein said variant comprisesa modification selected from the group consisting between One and fivedeletions at positions 34-39 and contains a Lys at position 40 having alipophilic substituent; or (c) GLP-1 (7-36) (SEQ ID NO: 114) or GLP-1(7-37) (SEQ ID NO: 124) having at least one modification selected fromthe group consisting of: (i) substitution of D-alanine, glycine oralpha-amino isobutyric acid (Aib) for alanine at position 8 and (ii) alipophilic substituent; and Z, a peptide sequence of 4-20 amino acidunits covalently bound to X, wherein each amino acid unit in saidpeptide sequence Z is selected from the group consisting of Ala, Leu,Ser, Thr, Tyr, Asn, Gin, Asp, Glu, Lys, Arg, His, Met, Orn, and aminoacid units of the general formula I

—NH—C(R¹)(R²)—C(═O)—  (I)

[0060] wherein R¹ and R² are selected from the group consisting ofhydrogen, C₁₋₆-alkyl, phenyl, and phenyl-methyl, wherein C₁₋₆-alkyl isoptionally substituted with from one to three substituents selected fromhalogen, hydroxy, amino, cyano, nitro, sulfono, and carboxy, and phenyland phenyl-methyl is optionally substituted with from one to threesubstituents selected from C₁₋₆-alkyl, C₂₋₆-alkenyl, halogen, hydroxy,amino, cyano, nitro, sulfono, and carboxy, or R¹ and R² together withthe carbon atom to which they are bound form a cyclopentyl, cyclohexyl,or cycloheptyl ring, e.g. 2,4-diaminobutanoic acid and2,3-diaminopropanoic acid. Preferably, X binds to a GLP-1 receptor anddoes not include exendin-4 or exendin-3.

[0061] Z is typically a peptide sequence of 4-20 amino acid residues,e.g., in the range of 4-15, more preferably in the range of 4-10 inparticular in the range of 4-7 amino acid residues, e.g., of 4, 5, 6, 7,8 or 10 amino acid residues, where 6 amino acid residues are preferred.Preferably, Z contains at least one Lys residue. In a preferredembodiment of the invention each of the amino acid residues in thepeptide sequence Z are independently selected from the group sonsistingof Ala, Leu, Ser, Thr, Tyr, Asn, Gin, Asp, Glu, Lys, Arg, His, Met, Orn,diaminobutanoic acid and diaminopropanoic acid. Preferably, the aminoacid residues are selected from Glu, Lys, and Met, especially Lys, orthe amino acid residues are selected from the group consisting of Asn,Glu and Lys. The above-mentioned amino acids may have either D- orL-configuration, but preferably the above-mentioned amino acids have anL-configuration. In a preferred embodiment of the invention Z containsat least 1 lysine residue or when Z is attached via a peptide bond tothe N-terminal of said peptide X then Z has an amino acid sequenceselected from the group consisting of Asn-(Glu)n wherein n is an integerfrom 3 to 7.

[0062] Thus, illustrative examples of the peptide sequence Z are:

[0063] Lys-Lys-Lys-Lys (SEQ ID NO: 1), Xaa-Lys-Lys-Lys, Lys-Xaa-Lys-Lys,Lys-Lys-Xaa-Lys, Lys-Lys-Lys-Xaa, Xaa-Xaa-Lys-Lys, Xaa-Lys-Xaa-Lys,Xaa-Lys-Lys-Xaa, Lys-Xaa-Xaa-Lys, Lys-Xaa-Lys-Xaa, Lys-Lys-Xaa-Xaa,Xaa-Xaa-Xaa-Lys, Xaa-Xaa-Lys-Xaa, Xaa-Lys-Xaa-Xaa, Lys-Xaa-Xaa-Xaa,Xaa-Xaa-Xaa-Xaa (SEQ ID NO:2), Lys-Lys-Lys-Lys-Lys (SEQ ID NO:3),Xaa-Lys-Lys-Lys-Lys (SEQ ID NO:4), Lys-Xaa-Lys-Lys-Lys (SEQ ID NO:5),Lys-Lys-Xaa-Lys-Lys (SEQ ID NO:6), Lys-Lys-Lys-Xaa-Lys (SEQ ID NO:7),Lys-Lys-Lys-Lys-Xaa, Xaa-Xaa-Lys-Lys-Lys, Xaa-Lys-Xaa-Lys-Lys,Xaa-Lys-Lys-Xaa-Lys, Xaa-Lys-Lys-Lys-Xaa, Lys-Xaa-Xaa-Lys-Lys,Lys-Xaa-Lys-Xaa-Lys, Lys-Xaa-Lys-Lys-Xaa, Lys-Lys-Xaa-Xaa-Lys,Lys-Lys-Xaa-Lys-Xaa, Lys-Lys-Lys-Xaa-Xaa, Lys-Lys-Xaa-Xaa-Xaa,Lys-Xaa-Lys-Xaa-Xaa, Lys-Xaa-Xaa-Lys-Xaa, Lys-Xaa-Xaa-Xaa-Lys,Xaa-Lys-Lys-Xaa-Xaa, Xaa-Lys-Xaa-Xaa-Lys, Xaa-Xaa-Lys-Lys-Xaa,Xaa-Xaa-Lys-Xaa-Lys, Xaa-Xaa-Xaa-Lys-Lys, Lys-Xaa-Xaa-Xaa-Xaa,Xaa-Lys-Xaa-Xaa-Xaa, Xaa-Xaa-Lys-Xaa-Xaa, Xaa-Xaa-Xaa-Lys-Xaa,Xaa-Xaa-Xaa-Xaa-Lys, Xaa-Xaa-Xaa-Xaa-Xaa (SEQ ID NO:8),Lys-Lys-Lys-Lys-Lys-Lys (SEQ ID NO:9), Xaa-Lys-Lys-Lys-Lys-Lys (SEQ IDNO:10), Lys-Xaa-Lys-Lys-Lys-Lys (SEQ ID NO:11), Lys-Lys-Xaa-Lys-Lys-Lys(SEQ ID NO:12), Lys-Lys-Lys-Xaa-Lys-Lys (SEQ ID NO:13),Lys-Lys-Lys-Lys-Xaa-Lys (SEQ ID NO:14), Lys-Lys-Lys-Lys-Lys-Xaa (SEQ IDNO:15), Xaa-Xaa-Lys-Lys-Lys-Lys (SEQ ID NO:16), Xaa-Lys-Xaa-Lys-Lys-Lys(SEQ ID NO:17), Xaa-Lys-Lys-Xaa-Lys-Lys (SEQ ID NO:18),Xaa-Lys-Lys-Lys-Xaa-Lys (SEQ ID NO:19), Xaa-Lys-Lys-Lys-Lys-Xaa (SEQ IDNO:20), Lys-Xaa-Xaa-Lys-Lys-Lys (SEQ ID NO:21), Lys-Xaa-Lys-Xaa-Lys-Lys(SEQ ID NO:22), Lys-Xaa-Lys-Lys-Xaa-Lys (SEQ ID NO:23),Lys-Xaa-Lys-Lys-Lys-Xaa (SEQ ID NO:24), Lys-Lys-Xaa-Xaa-Lys-Lys (SEQ IDNO:25), Lys-Lys-Xaa-Lys-Xaa-Lys (SEQ ID NO:26), Lys-Lys-Xaa-Lys-Lys-Xaa(SEQ ID NO:27), Lys-Lys-Lys-Xaa-Xaa-Lys (SEQ ID NO:28),Lys-Lys-Lys-Xaa-Lys-Xaa (SEQ ID NO:29), Lys-Lys-Lys-Lys-Xaa-Xaa,Xaa-Xaa-Xaa-Lys-Lys-Lys, Xaa-Xaa-Lys-Xaa-Lys-Lys,Xaa-Xaa-Lys-Lys-Xaa-Lys, Xaa-Xaa-Lys-Lys-Lys-Xaa,Xaa-Lys-Xaa-Xaa-Lys-Lys, Xaa-Lys-Xaa-Lys-Xaa-Lys,Xaa-Lys-Xaa-Lys-Lys-Xaa, Xaa-Lys-Lys-Xaa-Xaa-Lys,Xaa-Lys-Lys-Xaa-Lys-Xaa, Xaa-Lys-Lys-Lys-Xaa-Xaa,Lys-Lys-Lys-Xaa-Xaa-Xaa, Lys-Lys-Xaa-Lys-Xaa-Xaa,Lys-Lys-Xaa-Xaa-Lys-Xaa, Lys-Lys-Xaa-Xaa-Xaa-Lys,Lys-Xaa-Lys-Lys-Xaa-Xaa, Lys-Xaa-Lys-Xaa-Lys-Xaa,Lys-Xaa-Lys-Xaa-Xaa-Lys, Lys-Xaa-Xaa-Lys-Lys-Xaa,Lys-Xaa-Xaa-Lys-Xaa-Lys, Lys-Xaa-Xaa-Xaa-Lys-Lys,Lys-Lys-Xaa-Xaa-Xaa-Xaa, Lys-Xaa-Lys-Xaa-Xaa-Xaa,Lys-Xaa-Xaa-Lys-Xaa-Xaa-Lys, Lys-Xaa-Xaa-Xaa-Lys-Xaa-Lys,Lys-Xaa-Xaa-Xaa-Xaa-Lys-Lys, Xaa-Lys-Lys-Xaa-Xaa-Xaa,Xaa-Lys-Xaa-Lys-Xaa-Xaa, Xaa-Lys-Xaa-Xaa-Lys-Xaa,Xaa-Lys-Xaa-Xaa-Xaa-Lys, Xaa-Xaa-Lys-Lys-Xaa-Xaa,Xaa-Xaa-Lys-Xaa-Lys-Xaa, Xaa-Xaa-Lys-Xaa-Xaa-Lys,Xaa-Xaa-Xaa-Lys-Lys-Xaa, Xaa-Xaa-Xaa-Lys-Xaa-Lys,Xaa-Xaa-Xaa-Xaa-Lys-Lys, Lys-Xaa-Xaa-Xaa-Xaa-Xaa,Xaa-Lys-Xaa-Xaa-Xaa-Xaa, Xaa-Xaa-Lys-Xaa-Xaa-Xaa,Xaa-Xaa-Xaa-Lys-Xaa-Xaa, Xaa-Xaa-Xaa-Xaa-Lys-Xaa,Xaa-Xaa-Xaa-Xaa-Xaa-Lys, Xaa-Xaa-Xaa-Xaa-Xaa-Xaa,Lys-Lys-Lys-Lys-Lys-Lys-Lys (SEQ ID NO:30), Xaa-Lys-Lys-Lys-Lys-Lys-Lys(SEQ ID NO:31), Lys-Xaa-Lys-Lys-Lys-Lys-Lys (SEQ ID NO:32),Lys-Lys-Xaa-Lys-Lys-Lys-Lys (SEQ ID NO:33), Lys-Lys-Lys-Xaa-Lys-Lys-Lys(SEQ ID NO:34), Lys-Lys-Lys-Lys-Xaa-Lys-Lys (SEQ ID NO:35),Lys-Lys-Lys-Lys-Lys-Xaa-Lys (SEQ ID NO:36), Lys-Lys-Lys-Lys-Lys-Lys-Xaa(SEQ ID NO:37), Xaa-Xaa-Lys-Lys-Lys-Lys-Lys (SEQ ID NO:38),Xaa-Lys-Xaa-Lys-Lys-Lys-Lys (SEQ ID NO:39), Xaa-Lys-Lys-Xaa-Lys-Lys-Lys(SEQ ID NO:40), Xaa-Lys-Lys-Lys-Xaa-Lys-Lys (SEQ ID NO:41),Xaa-Lys-Lys-Lys-Lys-Xaa-Lys (SEQ ID NO:42), Lys-Xaa-Xaa-Lys-Lys-Lys-Lys(SEQ ID NO:43), Lys-Xaa-Lys-Xaa-Lys-Lys-Lys (SEQ ID NO:44),Lys-Xaa-Lys-Lys-Xaa-Lys-Lys (SEQ ID NO:45), Lys-Xaa-Lys-Lys-Lys-Xaa-Lys(SEQ ID NO:46), Lys-Lys-Xaa-Xaa-Lys-Lys-Lys (SEQ ID NO:47),Lys-Lys-Xaa-Lys-Xaa-Lys-Lys (SEQ ID NO:48), Lys-Lys-Xaa-Lys-Lys-Xaa-Lys(SEQ ID NO:49), Lys-Lys-Lys-Xaa-Xaa-Lys-Lys (SEQ ID NO:50),Lys-Lys-Lys-Xaa-Lys-Xaa-Lys (SEQ ID NO:51), Lys-Lys-Lys-Lys-Xaa-Xaa-Lys(SEQ ID NO:52), Xaa-Xaa-Xaa-Lys-Lys -Lys-Lys (SEQ ID NO:53),Xaa-Xaa-Lys-Xaa-Lys-Lys-Lys (SEQ ID NO:54), Xaa-Xaa-Lys-Lys-Xaa-Lys-Lys(SEQ ID NO:55), Xaa-Xaa-Lys-Lys-Lys-Xaa-Lys (SEQ ID NO:56),Xaa-Lys-Xaa-Xaa-Lys-Lys-Lys (SEQ ID NO:57), Xaa-Lys-Xaa-Lys-Xaa-Lys-Lys(SEQ ID NO:58), Xaa-Lys-Xaa-Lys-Lys-Xaa-Lys (SEQ ID NO:59),Xaa-Lys-Lys-Xaa-Xaa-Lys-Lys (SEQ ID NO:60), Xaa-Lys-Lys-Xaa-Lys-Xaa-Lys(SEQ ID NO:61), Xaa-Lys-Lys-Lys-Xaa-Lys-Xaa (SEQ ID NO:62),Xaa-Lys-Lys-Xaa-Lys-Lys-Xaa (SEQ ID NO:63), Xaa-Lys-Xaa-Lys-Lys-Lys-Xaa(SEQ ID NO:64), Xaa-Lys-Lys-Lys-Xaa-Xaa-Lys (SEQ ID NO:65),Lys-Xaa-Lys-Lys-Lys-Xaa-Xaa (SEQ ID NO:66), Xaa-Lys-Lys-Lys-Lys-Xaa-Xaa(SEQ ID NO:67), Xaa-Lys-Lys-Lys-Xaa-Lys-Xaa (SEQ ID NO:68),Xaa-Lys-Lys-Lys-Xaa-Xaa-Lys-(SEQ ID NO:0.69),Lys-Lys-Lys-Lys-Xaa-Xaa-Xaa (SEQ ID NO:70), Lys-Lys-Lys-Xaa-Xaa-Xaa-Lys(SEQ ID NO:71), Lys-Lys-Lys-Xaa-Lys-Xaa-Xaa (SEQ ID NO:72),Lys-Lys-Xaa-Lys-Lys-Xaa-Xaa (SEQ ID NO:73), Lys-Lys-Xaa-Xaa-Lys-Xaa-Lys(SEQ ID NO:74), Lys-Lys-Xaa-Xaa-Xaa-Lys-Lys (SEQ ID NO:75),Lys-Lys-Xaa-Lys-Lys-Xaa-Xaa (SEQ ID NO:76), Lys-Xaa-Lys-Lys-Xaa-Xaa-Lys(SEQ ID NO:77), Lys-Xaa-Lys-Xaa-Lys-Xaa-Lys (SEQ ID NO:78),Lys-Xaa-Lys-Xaa-Xaa-Lys-Lys (SEQ ID NO:79), Lys-Xaa-Xaa-Lys-Lys-Xaa-Lys(SEQ ID NO:80), Lys-Xaa-Xaa-Lys-Xaa-Lys-Lys (SEQ ID NO:81),Lys-Xaa-Xaa-Xaa-Lys-Lys-Lys (SEQ ID NO:82), Lys-Lys-Xaa-Xaa-Xaa-Xaa-Lys,Lys-Xaa-Lys-Xaa-Xaa-Xaa-Lys, Lys-Xaa-Xaa-Lys-Xaa-Xaa-Lys,Lys-Xaa-Xaa-Xaa-Lys-Xaa-Lys, Lys-Xaa-Xaa-Xaa-Xaa-Lys-Lys,Xaa-Lys-Lys-Xaa-Xaa-Xaa-Lys, Xaa-Lys-Xaa-Lys-Xaa-Xaa-Lys,Xaa-Lys-Xaa-Xaa-Lys-Xaa-Lys, Xaa-Lys-Xaa-Xaa-Xaa-Lys-Lys,Xaa-Xaa-Lys-Lys-Xaa-Xaa-Lys, Xaa-Xaa-Lys-Xaa-Lys-Xaa-Lys,Xaa-Xaa-Lys-Xaa-Xaa-Lys-Lys, Xaa-Xaa-Xaa-Lys-Lys-Xaa-Lys,Xaa-Xaa-Xaa-Lys-Xaa-Lys-Lys, Xaa-Xaa-Xaa-Xaa-Lys-Lys-Lys,Lys-Xaa-Xaa-Xaa-Xaa-Xaa-Lys, Xaa-Lys-Xaa-Xaa-Xaa-Xaa-Lys,Xaa-Xaa-Lys-Xaa-Xaa-Xaa-Lys, Xaa-Xaa-Xaa-Lys-Xaa-Xaa-Lys,Xaa-Xaa-Xaa-Xaa-Lys-Xaa-Lys, Xaa-Xaa-Xaa-Xaa-Xaa-Lys-Lys,Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Lys, Lys-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa,Xaa-Xaa-Xaa-Xaa-Xaa-Lys-Xaa, Xaa-Lys-Xaa-Xaa-Xaa-Xaa-Xaa,Xaa-Xaa-Lys-Xaa-Xaa-Xaa, Xaa-Xaa-Xaa-Xaa-Lys-Xaa,Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa, wherein each Xaa is independently selectedfrom the group consisting of Ala, Leu, Ser, Thr, Tyr, Asn, Gin, Asp,Glu, Arg, His, Met, Orn, and amino acids of the formula I as definedherein, e.g., Dbu or Dpr.

[0064] As indicated above, the amino acid residues of Z may of courseall be different or all be identical. However, in interestingembodiments of the present invention, the amino acid residues in Z areselected from two or three different amino acids, or are identical aminoacids. Examples of suitable peptide sequences, wherein the amino acidresidues in Z are identical are e.g., (Lys)_(n), wherein n is an integerin the range from 4 to 15, preferably in the range from 4 to 10, such asin the range from 4 to 8, e.g., in the range from about 4 to 7, e.g.,Lys₄ (SEQ ID NO:1), Lys₅ (SEQ ID NO:2), Lys₆ (SEQ ID NO:8), Lys₇ (SEQ IDNO:30). Preferred is (Lys)₆ bound via a peptide bond to the C-terminalof X.

[0065] Examples of suitable peptide sequences, wherein the amino acidresidues in Z are selected from about two different amino acids aree.g., (Lys-Xaa)_(m) or (Xaa-Lys)_(m), wherein m is an integer in therange from about 2 to 7, preferably in the range from 2 to 5, such as inthe range from 2 to 4, e.g., 3, and Xaa is independently selected fromthe group consisting of Ser, Thr, Tyr, Asn, Gin, Asp, Glu, Arg, His,Orn, 2,4-diaminobutanoic acid, 2,3-diaminopropanoic acid and Met. Morepreferably such peptide sequences are e.g., (Lys-Xaa)₃ or (Xaa-Lys)₃,wherein Xaa is as defined above, such as (Lys-Glu)₃ (SEQ ID NO:83) or(Glu-Lys)₃ (SEQ ID NO:84). Other examples of suitable peptide sequences,wherein the amino acid residues in Z are selected from about two aminoacid residues are e.g., Lys_(p)-Xaa_(q) or Xaa_(p)-Lys_(q), wherein pand q are integers in the range from 1 to 14, with the proviso that p+qis in the range from 4 to 15, preferably in the range from 4 to 10, suchas in the range from 4 to 8, e.g., in the range from 4 to 6, e.g., 4, 5or 6, and Xaa is independently selected from the group consisting ofSer, Thr, Tyr, Asn, Gin, Asp, Glu, Arg, His and Met. More preferablysuch peptide sequences are e.g., Lys₃-Xaa₃ or Xaa₃-Lys₃, wherein Xaa isas defined above, such as Lys₃-Glu₃ (SEQ ID NO:85) or Glu₃-Lys₃ (SEQ IDNO:86). More preferred Z sequences consists of a sequence of amino acidresidues selected from Asn and Gln together with 4-7 amino acid residuesselected from Glu and Asp, such as Asn-(Glu)₅(SEQ ID NO: 141),Asn-(Glu)₆(SEQ ID NO: 142), Gln-(Glu)₅(SEQ ID NO: 143), Asn-(Asp)₅(SEQID NO: 144), and Gln-(Asp)₅(SEQ ID NO: 145), which is the N-terminalpart of the peptide conjugate of the invention.

[0066] Examples of suitable peptide sequences, wherein the amino acidresidues in Z are selected from three different amino acids are e.g.,Xaa¹-(Lys)_(x)-(Xaa²)_(y), Xaa¹-(Xaa²)_(x)-(Lys)_(y),(Lys)_(x)-(Xaa²)_(y)-Xaa¹, (Xaa¹)_(x)-(Lys)_(y)-Xaa²,(Lys)_(x)-Xaa¹-(Xaa²)_(y), (Xaa¹)_(x)-Xaa²-(Lys)_(y), Xaa¹-Lys-Xaa²-Lys,Xaa¹-Lys-Xaa²-Lys-Xaa², Xaa¹-Lys-Xaa²-Lys-Xaa²-Lys, Xaa¹-Xaa²-Lys-Xaa²,Xaa¹-Xaa²-Lys-Xaa²-Lys, Xaa¹-Xaa²-Lys-Xaa²-Lys-Xaa², Lys-Xaa²-Lys-Xaa¹,Lys-Xaa²-Lys-Xaa²-Xaa¹, Lys-Xaa²-Lys-Xaa²-Lys-Xaa¹, Xaa²-Lys-Xaa²-Xaa¹,Xaa²-Lys-Xaa²-Lys-Xaa¹, Xaa²-Lys-Xaa¹-Lys-Xaa²-Xaa¹, etc., wherein x andy are integers in the range from about 1 to 5 with the proviso that x+yis at the most 6, and Xaa¹ and Xaa² is independently selected from aboutthe group consisting of Ala, Leu, Ser, Thr, Tyr, Asn, Gin, Asp, Glu,Arg, His, Met, Orn, 2,3-diaminopropanoic acid, 2,4-diaminobutanoic acidand amino acids of the formula I as defined herein.

[0067] In preferred embodiments of the invention the ratio between theminimum effective oral dose of said peptide conjugate and the minimumeffective dose of the peptide, X is at least 1:5.

[0068] A most preferred embodiment of the invention is directed to anovel peptide conjugate comprising a peptide X being an agonist of GLP-1and/or exendin-4 activity selected from the group consisting of

[0069] des Pro³⁶-exendin-4(1-39)-NH₂ (SEQ ID NO:101),

[0070] des Pro³⁶-exendin-4(1-40)-NH₂(SEQ ID NO: 139),

[0071] des Pro³⁶-des Pro³⁷-exendin-4 (1-39)-NH₂ (SEQ ID NO: 130),

[0072] des Pro³⁶-des Pro³⁷-des Pro³ ^(₈) -exendin-4(1-39)-NH₂(SEQ ID NO:132),

[0073] des Pro³⁶-des Pro³⁷-des Pro³ ^(₈) -exendin-4 (1-40)-NH₂(SEQ IDNO: 140),

[0074] des Ala³⁵-exendin-4(1-39)-NH₂ (SEQ ID NO:105),

[0075] des Gly³⁴-exendin-4(1-39)-NH₂ (SEQ ID NO:106),

[0076] des Gly³⁴-(Lys⁴⁰(palmitoyl))exendin-4(1-39)-NH₂ (SEQ ID NO:108),

[0077] des Ala³⁵(Lys⁴⁰(palmitoyl))exendin-4(139)-NH₂ (SEQ ID NO:109),

[0078] des Pro³⁶-(Lys⁴⁰(palmitoyl))exendin-4(1-39)-NH₂ (SEQ ID NO:110),

[0079] Compound (iii) Gly⁸-GLP-1(7-36)-NH₂(SEQ ID NO: 87),Gly⁸-GLP-1(7-37) (SEQ ID NO: 123), andGly⁸-GLP-1(7-36)-Lys³⁷(palmitoyl)-NH₂(SEQ ID NO: 147), and beingC-terminally bound via a peptide bond to a peptide sequence Z selectedfrom the group consisting of (Lys)n where n is an integer from 4 to 8,preferably n is 6.

[0080] It should be understood that the peptide conjugates of theinvention might also be in the preferred amide (NH₂) or in the free acid(OH) form or in the form of a salt thereof. Exemplary peptide conjugatesof the invention are

[0081] Gly⁸-GLP-1 (7-36)-Lys₆-NH₂ (SEQ ID NO:88),

[0082] (Gly⁸,Lys³⁷(palmitoyl)-GLP-1(7-36)(Human)-Lys₇-NH₂ (SEQ IDNO:89),

[0083] des Ser³⁹-exendin-4(1-39)-(Lys)₆-NH₂ (SEQ ID NO:91),

[0084] exendin-4(1-39)-Lys₆-NH₂ (SEQ ID NO:92),

[0085] des Pro³⁶-exendin-4 (1-39)-Lys₆-NH₂ (SEQ ID NO:93),

[0086] des Ala³⁵exendin-4(1-39)-Lys₆-NH₂ (SEQ ID NO:94),

[0087] des Gly³⁴exendin-4(1-39)-Lys₆-NH₂ (SEQ ID NO:95),

[0088] des Ser³⁹-(Lys⁴⁰(palmitoyl))exendin-4 (1-39)-Lys₇-NH₂ (SEQ IDNO:96),

[0089] des Gly³⁴-(Lys⁴⁰ (palmitoyl))exendin-4(1-39)-Lys₇-NH₂ (SEQ IDNO:97),

[0090] des Ala³⁵-(Lys⁴⁰(palmitoyl))exendin-4(1-39)-Lys₇-NH₂ (SEQ IDNO:98),

[0091] des Pro³⁶(Lys⁴⁰(palmitoyl))exendin-4(1-39)-Lys₇-NH₂ (SEQ IDNO:99),

[0092] Lys⁴⁰(palmitoyl)exendin-4(1-39)-Lys₇-NH₂(SEQ ID NO:100),

[0093] des Pro³⁶, Pro³⁷-exendin-4(1-39)-Lys₇-NH₂ (SEQ ID NO:131),

[0094] Lys₆-des Pro³⁶, Pro³⁷, Pro³ ^(₈) -exendin-4(1-39)-NH₂(SEQ IDNO:134),

[0095] Asn(Glu)₅-des Pro³⁶, Pro³⁷, Pro³ ^(₈) -exendin-4(1-39)-NH₂(SEQ IDNO: 137),

[0096] Lys₆-des Pro³⁶, Pro³⁷, Pro³ ^(₈) -exendin-4(1-39)-Lys₆-NH₂(SEQ IDNO:135),

[0097] Asn(Glu)₅-des Pro³⁶, Pro³⁷, Pro³ ^(₈) -exendin-4(1-39)-NH₂(SEQ IDNO:136),

[0098] des Pro³⁶, Pro³⁷, Pro³ ^(₈) -exendin-4(1-39)-Lys₆-NH₂SEQ IDNO:133)

[0099] Ser⁸GLP-1(7-36)-Lys₆-NH₂(SEQ ID NO:115),

[0100] Aib⁸-GLP-1 (7-36)-Lys₆-NH₂ (SEQ ID NO: 116),

[0101] Lys₆-Gly⁸-GLP-1(7-36)-Lys₆-NH₂ (SEQ ID NO: 118),

[0102] Lys₆-Gly⁸-GLP-1(7-36)-NH₂ (SEQ ID NO: 119),

[0103] (Gly⁸,Lys² ⁶ (palmitoyl)-GLP-1 (7-36)(Human)-Lys₆-NH₂ (SEQ ID NO:103),

[0104] (Gly⁸ Lys³⁴(palmitoyl)-GLP-1(7-36)(Human)-Lys₆-NH₂ (SEQ ID NO:90),

[0105] Gly⁸-GLP-1(7-36)-Lys₈-NH₂(SEQ ID NO: 120),

[0106] Gly⁸-GLP-1 (7-36)-Lys₁₀-NH₂(SEQ ID NO: 121),

[0107] Gly⁸-GLP-1 (7-37)-Lys₆-NH₂(SEQ ID NO: 122),

[0108] and the free acid thereof and a pharmaceutically acceptable saltthereof.

[0109] Among the Preferred conjugates are

[0110] des Pro³⁶-exendin-4(1-39)-Lys₆-NH₂ (SEQ ID NO:93),

[0111] Gly⁸-GLP-1 (7-36)-Lys₆-NH₂ (SEQ ID NO:88),

[0112] des Pro³⁶, Pro³⁷, Pro³⁸-exendin-4(1-39)-Lys₆-NH₂(SEQ ID NO: 133),and

[0113] their salts as defined herein.

[0114] In a most specific embodiment, the conjugates are selected fromthe group consisting of Gly⁸-GLP-1-(7-36)(Human)-NH₂(SEQ ID NO: 87),Gly⁸-GLP-1-(7-36)(Human)-Lys₆-NH₂(SEQ ID NO: 88),Gly⁸Lys³⁷(palmitoyl)-GLP-1-(7-36)(Human)-Lys₇-NH₂(SEQ ID NO: 89),Gly⁸Lys³⁴(palmitoyl)-GLP-1-(7-36)(Human)-Lys₆-NH₂(SEQ ID NO: 90), desSer³⁹-exendin-4(1-39)-Lys₆-NH₂(SEQ ID NO: 91),exendin-4(1-39)-Lys₆-NH₂(SEQ ID NO: 92), desPro³⁶-exendin-4(1-39)-Lys₆-NH₂(SEQ ID NO: 93), des Ala35-exendin-4(1-39)-Lys₆-NH₂(SEQ ID NO: 94), desGly³⁴-exendin-4(1-39)-Lys₆-NH₂(SEQ ID NO: 95), desSer³⁹-(Lys⁴⁰(palmitoyl))exendin-4(1-39)-Lys₇-NH₂(SEQ ID NO: 96), desGly³⁴-(Lys⁴⁰ (palmitoyl))exendin-4(1-39)-Lys₇-NH₂(SEQ ID NO: 97), desAla³⁵-(Lys⁴⁰ (palmitoyl))exendin-4(1-39)-Lys₇-NH₂(SEQ ID NO: 98), desPro³⁶-(Lys₄₀ (palmitoyl))exendin-4(1-39)-Lys₇-NH₂ (SEQ ID NO: 99) andLys₄₀ (palmitoyl)exendin-4(1-39)-Lys₇-NH₂(SEQ ID NO: 100).

[0115] The provision of the peptide conjugates of the present inventionenables blood glucose lowering peptides, such as GLP-1 and exendins andtheir active analogues to be administered orally. The herein preferredterminal peptide fragments Z are chosen so as to induce an alpha-helicalstructure to the peptide X without significantly affecting the desiredactivity of X. Said helical structure stabilises the peptide chain, e.g.againsts degradation, as evidenced by the increased half life of from 2to 3 times of the conjugated peptide compared to the unconjugatedpeptide, cf. table 5 below. The peptide sequence Z is the part of thepeptide conjugate responsible for introducing of a certain structureinto the molecule so that the minimum effective dose is lowered at leastfive fold. Preferably the minimum effective dose is lowered at least tenfold, more preferably 25 fold, even more preferably 40 fold, and mostpreferably 50 fold. Therefore, the present invention also relates to theuse of a peptide sequence (Z) as defined above for the preparation of asaid peptide conjugate as defined above.

[0116] Thus, the invention also relates to a novel peptide conjugatecomprising a peptide X as defined herein and wherein X reduces the bloodglucose level in a mammal where the ratio between the minimum effectiveoral dose of said peptide conjugate and the minimum effective oral doseof the peptide X is at least 1:5.

[0117] Specifically, the invention is directed to a method forstimulating insulin release in a mammal comprising administering aneffective insulinotropic amount of the peptide conjugate of the presentinvention, a method of lowering blood glucose level in a mammalcomprising administering an amount of the peptide conjugate of thepresent invention effective to lower blood glucose level in said mammal,a method of reducing gastric motility in a mammal in an amount of thepeptide conjugate of the present invention effective to reduce gastricmotility, a method of delaying gastric emptying in a mammal in an amountof the peptide conjugate of the present invention effective to delaygastric emptying, a method of inhibiting food uptake in a mammal in anamount of the peptide conjugate of the present invention effective toinhibit food uptake and a method of lowering plasma lipid level in amammal comprising administering an amount of peptide conjugate of thepresent invention effective to lower plasma lipid level in said mammal.Specifically, the peptide conjugate of the present invention may be usedin treatment of diabetes type 1 or type 2, obesity, eating disorders,hyperglycemia, metabolic disorders, gastric disease and insulinresistance syndrome.

[0118] The present invention also relates to methods for the preparationof said peptide conjugate, by means of recombinant DNA technologycomprising the steps of (a) introducing a nucleic acid sequence encodingsaid conjugate into a host cell and (b) culturing said host cell and (c)isolating said conjugate from the culture or (a) culturing a recombinanthost cell comprising a nucleic acid sequence encoding said conjugateunder conditions permitting the production of said conjugate and (b)isolating said conjugate from the culture.

[0119] The method also relates to methods for the preparation of saidpeptide conjugate in which peptide X is obtained via recombinant DNAmethods by isolating said peptide. X is then conjugated to Z which isattached to a solid support or has been prepared by solid phasesynthetic methods. Furthermore, the invention relates to the preparationof the peptide conjugate of the present invention by peptide syntheticmethods. Furthermore, the invention relates to the preparation of thepeptide conjugate of the present invention by peptide synthetic methods.

[0120] The conjugates of the invention comprising an N-terminal sequenceof from 33 to 39, preferably from 36 to 38, amino acid residues having asubstantial homology to the native exendin-4 N-terminal sequence thoughtto be essential for receptor binding (insulinotropic activity) and aC-terminal sequence Z possess as a further advantage improved stabilitycompared to native exendins and C-terminally truncated forms of exendin.Likewise, the GLP-1 peptide conjugate Compound 4 shows improvedstability compared to the unconjugated Compound (iii).

[0121] Compositions

[0122] The invention also concerns a composition comprising the exendinvariant or the peptide conjugate of the present invention in combinationwith a physiologically acceptable carrier. Such compositions may be in aform adapted to oral, parenteral (including subcutaneous (s.c.),intravenous (i.v.), intramuscular (i.m.), epidural, direct brain andintraperitoneal (i.p.)), rectal, intratracheal, intranasal, dermal,vaginal, buccal, ocularly, or pulmonary administration, preferably in aform adapted to subcutaneous or oral administration, and suchcompositions may be prepared in a manner well-known to the personskilled in the art, e.g., as generally described in “Remington'sPharmaceutical Sciences”, 17. Ed. Alfonso R. Gennaro (Ed.), MarkPublishing Company, Easton, Pa., U.S.A., 1985 and more recent editionsand in the monographs in the “Drugs and the Pharmaceutical Sciences”series, Marcel Dekker. The compositions may appear in conventionalforms, for example, capsules, tablets, aerosols, topical applicationforms, liquid or semiliquid forms, such as solutions, suspensions,dispersions, emulsions, micelles or liposomes. Preferred are liquidcompositions suitable for s.c. administration. In a preferredembodiment, the compositions of the present invention are administeredsubcutaneously. In an alternative preferred embodiment, the compositionsof the present invention are administered orally, and in such cases onepreferred administration form is a tablet or capsule.

[0123] The pharmaceutical carrier or diluent employed may be aconventional solid or liquid carrier. Examples of solid carriers arelactose, terra alba, sucrose, cyclodextrin, talc, gelatin, agar, pectin,acacia, magnesium stearate, stearic acid ro lower alkyl ethers ofcellulose. Examples of liquid carriers are syrup, peanut oil, olive oil,phospholipids, sterols, fatty acids, fatty acid amines, polyoxyethylene,isotonic buffer solutions and water. Similarly, the carrier or diluentmay include any sustained release material known in the art, such asglyceryl monostearate or glyceryl distearate, alone or mixed with a wax.If a solid carrier is used for oral administration, the preparation maybe tabletted, placed in a hard gelatin capsule in powder or pellet formor it can be in the form of a troche or lozenge. The amount of solidcarrier will vary widely but will usually be from about about 25 mg toabout 1 g.

[0124] A typical tablet which may be prepared by conventional tablettingtechniques may contain:

[0125] Core: active compound (as free compound of the invention or saltthereof) 100 mg; colloidal silicon dioxide (Aerosil) 1.5 mg; cellulose,microcryst. (Avicel) 70 mg; modified cellulose gum (Ac-Di-Sol) 7.5 mg;magnesium stearate.

[0126] Coating: HPMC approx. 9 mg; *Mywacett 9-40T approx. 0.9 mg;*acylated monoglyceride used as plasticizer for film coating.

[0127] If a liquid carrier is used, the preparation may be in the formof a syrup, emulsion, soft gelatin capsule or sterile injectable liquidsuch as an aqueous or non-aqueous liquid suspension or solution.

[0128] For nasal administration, the preparation may contain a compoundof the present invention, preferably a conjugate, dissolved or suspendedin a liquid carrier, in particular, an aqueous carrier, for aerosolapplication. The carrier may contain additives such as solubilizingagents, e.g., propylene glycol, surfactants such as bile acid salts orpolyoxyethylene higher alcohol ethers, absorption enhancers such aslecithin (phosphatidylcholine) or cyclodextrin, or preservatives such asparabines.

[0129] The composition may also be in a form suited for local orsystemic injection or infusion and may, as such, be formulated withsterile water or an isotonic saline or glucose solution. Thecompositions may be sterilized by conventional sterilization techniqueswhich are well known in the art. The resulting aqueous solutions may bepackaged for use or filtered under aseptic conditions and lyophilized,the lyophilized preparation being combined with the sterile aqueoussolution prior to administration. Preferably, the formulation to be usedfor intravenous, subcutaneous and oral dosing will be a solution of theactive compound in buffer. The preparation may be produced immediatelybefore use from active drug substance and sterile buffer solution. Onepreferred method of sterilization may be by sterile filtration of asolution made immediately prior to use. The composition may containpharmaceutically acceptable auxiliary substances as required toapproximate physiological conditions, such as buffering agents, tonicityadjusting agents and the like, for instance sodium acetate, sodiumlactate, sodium chloride, potassium chloride, calcium chloride, etc.

[0130] The compounds of the invention possess valuable pharmacologicalproperties, e.g. stability towards proteolytic enzymes. In vitrostability studies with the present peptides and peptide conjugates inthe presence of selected proteolytic enzymes show increased half livesof the novel peptides compared to prior art peptides. Thus, thecompounds of the invention exhibit considerably extended duration ofaction in vivo compared to GLP-1 and other GLP-1 agonists. Furthermore,the compounds of the invention stimulate cAMP formation. This effect maybe demonstrated in a cAMP assay, e.g. as described in WO 98/08871.

[0131] The peptide compounds of the present invention are agonists ofGLP-1 activity and/or exendin-4 activity and improves blood glucosetolerance in diabetic mammals as determined by assays known in the artfor a particular peptide. Examples of such an assay are describedherein. Thus, the invention also concerns the exendin variants andpeptide conjugates as defined above for use in therapy, and the use ofthe peptide conjugates as defined above for the manufacture of apharmaceutical composition for use in therapy, e.g., in the treatment ofdiabetes type 1 or type 2, obesity, eating disorders and insulinresistance syndrome.

[0132] In specific embodiments, the exendin variants and peptideconjugates of the invention may be used to stimulate insulin release,lower blood glucose level, reduce gastric motility, delay gastricemptying, inhibit food uptake, e.g. by suppression of appetite, or lowerthe plasma lipid level in a vertebrate or a mammal. The novel compoundsof the invention may also be used generally in the treatment of diabetesmellitus associated with a risk for hyperglycemia, i.e. where insulinsensitivity is decreased with stress, myocardia infection, stroke andinfections, or in cases of insulin resistance during pregnancy. Thenovel compounds may also be used in the treatment of other types ofdiabetes, such as cases where diabetes may be secondary to otherendocrine diseases such as acromegaly, Cushing's syndrome,pheochromocytoma, glucagonoma, somatostatinoma, primary aldosteronism,or secondary to adminstration of certain hormones causing hyperglycemia,or secondary to certain drugs (antihypertensive drugs, thiazidediuretics, preparations containing estrogen, psychoactive drugs,sympathomimetic agents. Furthermore, the novel compounds of theinvention may be used generally in the treatment of diseases andconditions associated with a risk for hypoglycemia, i.e. whereendogenous glucose production is decreased, as following alcoholingestion, or in cases where the sensitivity to insulin is increased inpatients with hypopituitarism or primary adrenocortical insufficiency,or where insulin clearance is devreased as with progressive renalinsufficieny.

[0133] Other specific therapeutic uses are described in WO 99/40788(relating to the inotropic and diuretic effects of exendin and GLP-1) WO98/39022 (relating to a method of sedating a mammalian subject havingincreased activation of the central or peripheral nervous systemcomprising administering exendin or GLP-1 or an agonist of exendin orGLP-1 to the subject to produce a sedative or anxiolytic effect on thesubject), WO 93/18786 (relating to the treatment of diabetes using GLP-1(7-37) (SEQ ID NO: 124) or GLP-1(7-36)amide (SEQ ID NO: 114) in aregimen which additionally comprises treatment with an oralhypoglycaemic agent, such as sulfonylurea, producing a strongsynergistic effect), WO 98/19698 (relating to the use of analogs for theregulation of obesity), WO 98/08531 (relating to the use of GLP-1 oranalogs in a method of reducing mortality and morbidity after myocardialinfarction), WO 98/08873 (relating to the use of GLP-1 or analogs in amethod of attenuating post-surgical catabolic changes and hormonalresponses to stress). Besides, the compounds of the invention aresuitable in a combination therapy with other antidiabetic agents, suchas insulin, metformin, sulfonyl ureas and thiazolidinediones, or incombination therapy with other antiobesity agents, such as leptin,dexphenfluramine, amphetamin etc.

[0134] Definitions

[0135] A “peptide” as used herein is any compound produced by amideformation between a carboxyl group of one amino acid and an amino groupof another. The amide bonds in peptides may be called peptide bonds. Theword peptide usually applies to compounds whose amide bonds are formedbetween C-1 of one amino acid and N-2 of another (sometimes calledeupeptide bonds), but it includes compounds with residues linked byother amide bonds (sometimes called isopeptide bonds). Peptides withfewer than about 10-20 residues may also be called oligopeptides; thosewith more, polypeptides. Polypeptides of specific sequence of more thanabout 50 residues are usually known as proteins. A “natural polypeptidesequence” as used herein refers to a polypeptide sequence consisting ofnatural L-amino acid residues and which is capable of being expressed bya recombinant host cell. The X compounds herein are all peptidesequences of 40 amino acid residues or less.

[0136] “GLP-1” as used herein includes GLP-1(7-37)-OH(SEQ ID NO: 124),GLP-1(7-37)-NH₂(SEQ ID NO: 124), GLP-1(7-36)-OH(SEQ ID NO: 114), andGLP-1(7-36)-NH₂(SEQ ID NO: 114).

[0137] “Agonist” refers to an endogenous substance or a drug that caninteract with a receptor and initiate a physiological or apharmacological response characteristic of that receptor (contraction,relaxation, secretion, enzyme activation, etc.).

[0138] “Antagonist” refers to a drug or a compound that opposes thephysiological effects of another. At the receptor level, it is achemical entity that opposes the receptor-associated responses normallyinduced by another bioactive agent.

[0139] “Partial agonist” refers to an agonist which is unable to inducemaximal activation of a receptor population, regardless of the amount ofdrug applied. A “partial agonist” may be termed “agonist withintermediate intrinsic efficacy” in a given tissue. Moreover, a partialagonist may antagonize the effect of a full agonist that acts on thesame receptor.

[0140] “Receptor” refers to a molecule or a polymeric structure in or ona cell that specifically recognizes and binds a compound acting as amolecular messenger (neurotransmitter, hormone, lymphokine, lectin,drug, etc.).

[0141] By “exendin variant” of the present invention is to be understooda variant of a parent exendin peptide having at least about 90% homologyto exendin-4 and most preferably having at least about 95% homology toexendin-4(1-39) (SEQ ID NO: 102), which has exendin activity, e.g.,lowers the blood glucose level in a mammal and binds to a GLP-1receptor. “Exendin-4” as used herein refers to exendin-4(1-39) (SEQ IDNO: 102) the amino acid sequence of which is disclosed in U.S. Pat. No.5,424,286, SEQ ID NO:2, and exendin-4(1-40) (SEQ ID NO: 138) asdisclosed by Chen & Drucker in The Journal of Biological Chemistry, Vol.272, No. 7, pp.4108-15 which differs only in having glycine in position40 as C-terminal amino acid residue. The homology of the parent exendinis determined as the degree of identity between two protein sequencesindicating a derivation of the first sequence from the second. Thehomology may suitably be determined by means of computer programs knownin the art such as GAP provided in the GCG program package (ProgramManual for the Wisconsin Package, Version 8, August 1994, GeneticsComputer Group, 575 Science Drive, Madison, Wis., USA 53711) (Needleman,S. B. and Wunsch, C. D., (1970), J. Mol. Biol. 48:443-453). Thefollowing settings for polypeptide sequence comparison may be used: GAPcreation penalty of 3.0 and GAP extension penalty of 0.1.

[0142] “Salts” include pharmaceutically acceptable salts, such as acidaddition salts and basic salts. Examples of acid addition salts arehydrochloride salts, sodium salts, hydrobromide salts, etc. Examples ofbasic salts are salts where the cation is selected from alkali metals,such as sodium and potassium, alkaline earth metals, such as calcium,and ammonium ions ⁺N(R³)₃(R⁴), where R³ and R⁴ independently designatesoptionally substituted C₁₋₆-alkyl, optionally substituted C₂₋₆-alkenyl,optionally substituted aryl, or optionally substituted heteroaryl. Otherexamples of pharmaceutically acceptable salts are; e.g., those describedin “Remington's Pharmaceutical Sciences” 17. Ed. Alfonso R. Gennaro(Ed.), Mark Publishing Company, Easton, Pa., U.S.A., 1985 and morerecent editions, and in Encyclopedia of Pharmaceutical Technology.

[0143] Preparation of Variants and Conjugates

[0144] The exendin variants and the peptide conjugates of the inventionmay be prepared by methods known per se in the art. Thus, the variantsand the peptide sequences X and Z may be prepared by standardpeptide-preparation techniques such as solution synthesis orMerrifield-type solid phase synthesis. It is believed that the Boc(tert.butyloxycarbonyl) as well as the Fmoc(9-fluorenylmethyloxycarbonyl) strategies are applicable. In onepossible synthesis strategy, the peptide conjugates of the invention maybe prepared by solid phase synthesis by first constructing the peptidesequence Z using well-known standard protection, coupling anddeprotection procedures, thereafter sequentially coupling the peptidesequence X on Z in a manner similar to the construction of Z, andfinally cleaving off the entire peptide conjugate from the carrier. Thisstrategy yields a peptide conjugate, wherein the peptide sequence Z iscovalently bound to the peptide X at the C-terminal carbonyl function ofX. If the desired peptide conjugate, however, is a peptide conjugate,wherein two stabilising sequences Z are covalently and independentlybound to both the C- and the N-terminal of the peptide X, the abovestrategy is also applicable but, as will be understood by the personskilled in the art, before cleaving the off the C-terminal bound peptideconjugate from the solid support, it is necessary to sequentially couplethe second peptide sequence Z to the N-terminal of X in a manner similarto the procedure described above. This strategy may also be used toattach Z to the carbonyl function on the side chain of Glu or Asp. Apossible strategy for the preparation of peptide conjugates, wherein thepeptide sequence Z is covalently bound to the N-terminal nitrogen atomor covalently bound to the nitrogen atom on the side chain of Lys, Argor His of X is analogous with the method described above, i.e. saidpeptide conjugates may be prepared by solid phase synthesis by firstconstructing the peptide sequence X using well-known standardprotection, coupling and deprotection procedures, thereaftersequentially coupling the peptide sequence Z on X in a manner similar tothe construction of X, and finally cleaving off the entire peptideconjugate from the carrier. Another possible strategy is to prepare oneor both of the two sequences X and Z (or parts thereof) separately bysolution synthesis, solid phase synthesis, recombinant techniques, orenzymatic synthesis, followed by coupling of the two sequences bywell-known segment condensation procedures, either in solution or usingsolid phase techniques or a combination thereof. In one embodiment, Xmay be prepared by recombinant DNA methods and Z may be prepared bysolid phase synthesis. The conjugation of X and Z may be carried out byusing chemical ligation. This technique allows for the assembling oftotally unprotected peptide segments in a highly specific manner (Liu etal., 1996, J. Am. Chem. Soc. 118:307-312 and Dawson et al., 1996,226:776). The conjugation can also be performed by protease-catalysedpeptide bond formation, which offers a highly specific technique tocombine totally unprotected peptide segments via a peptide bond (W.Kullmann, 1987, Enzymatic Peptide Synthesis, CRC Press, Boca Raton,Fla., pp. 41-59).

[0145] Side chain derivatization of Lys, Arg, His, Trp, Ser, Thr, Cys,Tyr, Asp and Glu with the peptide sequence, Z, can be carried out bytraditional convergent peptide synthesis using suitable orthogonalprotecting schemes as known in the art, or by using the equally wellknown general solid phase method with suitable orthogonal removablechain protection.

[0146] Furthermore, it is envisaged that a combination of theabove-mentioned strategies may be especially applicable where a modifiedpeptide sequence, e.g., from a peptide X comprising isosteric bonds suchas reduced peptide bonds, is to be coupled to a peptide sequence Z. Inthis case, it may be advantageous to prepare the immobilised fragment ofZ by successive coupling of amino acids, and then couple a completepeptide sequence X (prepared in solution or fully or partially usingsolid phase techniques or by means of recombinant techniques) to thefragment.

[0147] Examples of suitable solid support materials (SSM) are e.g.,functionalised resins such as polystyrene, polyacrylamide,polydimethylacrylamide, polyethyleneglycol, cellulose, polyethylene,polyethyleneglycol grafted on polystyrene, latex, dynabeads, etc. Itshould be understood that it may be necessary or desirable that theC-terminal amino acid of the peptide sequence Z or the C-terminal aminoacid of the peptide X is attached to the solid support material by meansof a common linker such as 2,4-dimethoxy-4′-hydroxy-benzophenone,4-(4-hydroxy-methyl-3-methoxyphenoxy)-butyric acid,4-hydroxy-methyl-benzoic acid, 4-hydroxymethyl-phenoxyacetic acid,3-(4-hydroxymethylphenoxy)propionic acid, andp-[(R,S)-a[1-(9H-fluoren-9-yl)methoxyformamido]-2,4-dimethoxybenzyl]-phenoxy-aceticacid.

[0148] The variants and the peptide conjugates of the invention may becleaved from the solid support material by means of an acid such astrifluoracetic acid, trifluoromethanesulfonic acid, hydrogen bromide,hydrogen chloride, hydrogen fluoride, etc. optionally in combinationwith one or more “scavengers” suitable for the purpose, e.g.,ethanedithiol, triisopropylsilane, phenol, thioanisole, etc., or thepeptide conjugate of the invention may be cleaved from the solid supportby means of a base such as ammonia, hydrazine, an alkoxide, such assodium ethoxide, an hydroxide, such as sodium hydroxide, etc.

[0149] Thus, the present invention also relates to a method for thepreparation of a pharmacologically active peptide conjugate, wherein Zis covalently bound to X, preferably via a peptide bond. A method forthe preparation of a peptide conjugate of formula I (X-Z), comprises thesteps of:

[0150] a) coupling an amino acid or dipeptide having suitable protectinggroups, including an N-α-protecting group, in the activated form to animmobilised peptide sequence H-Z-SSM, thereby forming an immobilisedN-α-protected peptide fragment,

[0151] b) removing said N-α-protecting group, thereby forming animmobilised protected peptide fragment having an unprotected N-terminal,

[0152] c) coupling an additional amino acid or dipeptide having suitableprotecting groups including an N-α-protecting group in the carboxylactivated form to the N-terminal of the immobilised peptide fragment,and repeating the removal/coupling step procedure in step b) and c)until the desired peptide sequence X is obtained, and then

[0153] d) cleaving off the peptide conjugate from the solid supportmaterial.

[0154] A method for the preparation of a peptide conjugate of formula II(Z-X), comprises the steps of:

[0155] a) coupling an amino acid or dipeptide having suitable protectinggroups, including an N-α-protecting group, in the activated form to asolid support material (SSM), thereby forming an immobilised protectedamino acid or a protected dipeptide,

[0156] b) removing said N-α-protecting group, thereby forming animmobilised amino acid or peptide fragment having an unprotectedN-terminal,

[0157] c) coupling an additional amino acid or dipeptide having suitableprotecting groups, including an N-α-protecting group, in the carboxylactivated form to the N-terminal of the immobilised amino acid orpeptide fragment, and repeating the removal/coupling step procedure instep b) and c) until the desired peptide sequence X is obtained,

[0158] d) coupling an additional amino acid or dipeptide having suitableprotecting groups, including an N-α-protecting group, in the carboxylactivated form to the N-terminal of the immobilised peptide fragment,and repeating the removal/coupling step procedure in step b) and d)until the desired peptide sequence Z is obtained, and then

[0159] e) cleaving off the peptide conjugate from the solid supportmaterial.

[0160] Furthermore, a method for the preparation of a peptide conjugateof formula III (Z-X-Z), comprises the steps of:

[0161] a) coupling an amino acid or dipeptide having suitable protectinggroups, including an N-α-protecting group, in the carboxyl activatedform to an immobilised peptide sequence H-Z-SSM, thereby forming animmobilised N-α-protected peptide fragment,

[0162] b) removing said N-α-protecting group, thereby forming animmobilised peptide fragment having an unprotected N-terminal,

[0163] c) coupling an additional amino acid or dipeptide having suitableprotecting groups, including an N-α-protecting group, in the carboxylactivated form to the N-terminal of the immobilised peptide fragment,and repeating the removal/coupling step procedure in step b) and c)until the desired peptide sequence X is obtained, and then

[0164] d) coupling an additional amino acid or dipeptide having suitableprotecting groups, including an N-α-protecting group, in the carboxylactivated form to the N-terminal of the immobilised peptide fragment,and repeating the removal/coupling step procedure in step b) and d)until the desired peptide sequence Z is obtained, and then

[0165] e) cleaving off the peptide conjugate from the solid supportmaterial.

[0166] The coupling, removal and cleavage steps are performed by methodsknown to the person skilled in the art taking into consideration theprotection strategy and the selected solid phase material. In general,however, it is believed that the Boc (tert.butyloxycarbonyl) as well asthe Fmoc (9-fluorenylmethyloxycarbonyl) protection strategies areapplicable and that peptide bonds may be formed using the variousactivation procedures known to the person skilled in the art, e.g., byreacting a C-terminal activated derivative (acid halide, acid anhydride,activated ester e.g., HObt-ester, etc.) of the appropriate amino acid orpeptide with the amino group of the relevant amino acid or peptide asknown to a person skilled in peptide chemistry. Furthermore, it may benecessary or desirable to include side-chain protection groups whenusing amino acid residues carrying functional groups which are reactiveunder the prevailing conditions. The necessary protection scheme will beknown to the person skilled in the art (cf., e.g., M. Bodanszky and A.Bodanszky, “The Practice of Peptide Synthesis”, 2. Ed, Springer-Verlag,1994, J. Jones, “The Chemical Synthesis of Peptides”, Clarendon Press,1991, and Dryland et al., 1986, J. Chem. Soc., Perkin Trans. 1:125-137).

[0167] The peptides and peptide conjugates of the invention may also beprepared by means of recombinant DNA technology using general methodsand principles known to the person skilled in the art. A nucleic acidsequence encoding the peptides and peptide conjugates may be preparedsynthetically by established standard methods, e.g., the phosphoamiditemethod described by S. L. Beaucage and M. H. Caruthers, TetrahedronLetters 22, 1981, pp. 1859-1869, or the method described by Matthes etal., EMBO Journal 3, 1984, pp. 801-805. According to the phosphoamiditemethod, oligonucleotides are synthesized, e.g., in an automatic DNAsynthesizer, purified, annealed, ligated and cloned in suitable vectors.The techniques used to isolate or clone a nucleic acid sequence encodingpeptide X are known in the art and include isolation from genomic DNA,preparation from cDNA, or a combination thereof. The cloning of thenucleic acid sequences of the present invention from such genomic DNAcan be effected, e.g., by using the well known polymerase chain reaction(PCR) or antibody screening of expression libraries to detect cloned DNAfragments with shared structural features. See, e.g., Innis et al.,1990, A Guide to Methods and Application, Academic Press, New York.Other nucleic acid amplification procedures such as ligase chainreaction (LCR), ligated activated transcription (LAT) and nucleic acidsequence-based amplification (NASBA) may be used. It can then be ligatedto a nucleic acid sequence encoding Z.

[0168] The nucleic acid sequence encoding the peptides and peptideconjugates is then inserted into a recombinant expression vector whichmay be any vector which may conveniently be subjected to recombinant DNAprocedures. The choice of vector will often depend on the host cell intowhich it is to be introduced. Thus, the vector may be an autonomouslyreplicating vector, i.e., a vector which exists as an extrachromosomalentity, the replication of which is independent of chromosomalreplication, e.g., a plasmid. Alternatively, the vector may be onewhich, when introduced into a host cell, is integrated into the hostcell genome and replicated together with the chromosome(s) into which ithas been integrated.

[0169] In the vector, the nucleic acid sequence encoding the peptidesand peptide conjugates of the present invention should be operablyconnected to a suitable promoter sequence. The promoter may be anynucleic acid sequence which shows transcriptional activity in the hostcell of choice and may be derived from genes encoding proteins eitherhomologous or heterologous to the host cell. Examples of suitablepromoters for directing the transcription of the nucleic acid sequenceencoding said peptides and peptide conjugates in mammalian cells are theSV 40 promoter (Subramani et al., Mol. Cell Biol. 1, 1981, pp. 854-864),the MT-1 (metallothionein gene) promoter (Palmiter et al., Science 222,1983, pp. 809-814) or the adenovirus 2 major late promoter, a Roussarcoma virus (RSV) promoter, cytomegalovirus (CMV) promoter (Boshart etal., 1981, Cell 41:521-530) and a bovine papilloma virus promoter (BPV).A suitable promoter for use in insect cells is the polyhedrin promoter(Vasuvedan et al., FEBS Lett. 311, 1992, pp. 7-11).

[0170] Examples of suitable promoters for directing the transcription ofthe nucleic acid sequence encoding the peptides and peptide conjugates,especially in a bacterial host cell, are the promoters obtained from theE coli lac operon, the Streptomyces coelicolor agarase gene (dagA), theBacillus subtilis levansucrase gene (sacB), the Bacillus licheniformisalpha-amylase gene (amyL), the Bacillus stearothermophilus maltogenicamylase gene (amyM), the Bacillus amyloliqziefaciens alpha amylase gene(amyQ), the Bacillus licheniformis penicillinase gene (penP), theBacillis subtilis xylA and xylB genes, and the prokaryoticbeta-lactamase gene (Villa-Kamaroff et al., 1978, Proceedings of theNational Academy of Sciences USA 75:3727-3731), as well as the tacpromoter (DeBoer et al., 1983, Proceedings of the National Academy ofSciences USA 80:21 25). Further promoters are described in “Usefulproteins from recombinant bacteria” in Scientific American, 1980,242:74-94; and in Sambrook et al., 1989, supra. Examples of suitablepromoters for directing the transcription of the nucleic acid sequenceencoding the peptides and peptide conjugates in a filamentous fungalhost cell are promoters obtained from the genes encoding Aspergillusoryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillusniger neutral alpha-amylase, Aspergillus niger acid stablealpha-amylase, Aspergillius niger or Aspergillius avamori glucoamylase(glaA), Rhizomucor miehei lipase, Aspergllits oryzae alkaline protease,Aspergillus oryzae triose phosphate isomerase, Aspergillus nidulansacetamidase, Fusarium oxysporum trypsin-like protease (as described inU.S. Pat. No. 4,288,627, which is incorporated herein by reference), andhybrids thereof. Particularly preferred promoters for use in filamentousfungal host cells are the TAKA amylase, NA2-tpi (a hybrid of thepromoters from the genes encoding Aspergillus niger neutral a amylaseand Aspergillus oryzae triose phosphate isomerase), and glaA promoters.In a yeast host, useful promoters are obtained from the Saccharomycescerevisiae enolase (ENO-1) gene, the Saccharomyces cerevisiaegalactokinase gene (GAL 1), the Saccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase genes (ADH2/GAP),and the Saccharomyces cerevisiae 3-phosphoglycerate kinase gene. Otheruseful promoters for yeast host cells are described by Romanos et al.,1992, Yeast 8:423-488.

[0171] The nucleic acid sequence encoding said peptides and peptideconjugates may also be operably connected to a suitable terminator, suchas the human growth hormone terminator (Palmiter et al., op. cit.)Preferred terminators for filamentous fungal host cells are obtainedfrom the genes encoding Aspergillus oryzae TAKA amylase, Aspergillusniger glucoamylase, Aspergillus nidulans anthranilate synthase,Aspergillus niger alpha-glucosidase, and Fusarium oxysporum trypsin-likeprotease. Preferred terminators for yeast host cells are obtained fromthe genes encoding Saccharomyces cerevisiae enolase, Saccharomycescerevisiae cytochrome C (CYC1), or Saccharomyces cerevisiaeglyceraldehyde-3-phosphate dehydrogenase. Other useful terminators foryeast host cells are described by Romanos et al., 1992, supra.

[0172] The vector may further comprise elements such as polyadenylationsignals (e.g., from SV 40 or the adenovirus 5 Elb region),transcriptional enhancer sequences (e.g., the SV 40 enhancer) andtranslational enhancer sequences (e.g., the ones encoding adenovirus VARNAs). Furthermore, preferred polyadenylation sequences for filamentousfungal host cells are obtained from the genes encoding Aspergillusoryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillusnidulans anthranilate synthase, and Aspergillus niger alpha-glucosidase.Useful polyadenylation sequences for yeast host cells are described byGuo and Sherman, 1995, Molecular Cellular Biology 15:5983-5990.

[0173] The recombinant expression vector may further comprise a DNAsequence enabling the vector to replicate in the host cell in question.Examples of such a sequence (when the host cell is a mammalian cell) isthe SV 40 or polyoma origin of replication. Examples of bacterialorigins of replication are the origins of replication of plasmidspBR322, pUC19, pACYC177, pACYC184, pUB110, pE194, pTA1060, and pAMβ1.Examples of origin of replications for use in a yeast host cell are the2 micron origin of replication, the combination of CEN6 and ARS4, andthe combination of CEN3 and ARS1. The origin of replication may be onehaving a mutation to make its function temperature-sensitive in the hostcell (see, e.g., Ehrlich, 1978, Proc. Natl. Acad. Sci. USA 75:1433).

[0174] The vector may also comprise a selectable marker, e.g., a genethe product of which complements a defect in the host cell, such as thegene coding for dihydrofolate reductase (DHFR) or one which confersresistance to a drug, e.g., neomycin, geneticin, ampicillin, orhygromycin. Suitable markers for yeast host cells are ADE2, HIS3, LEU2,LYS2, MET3, TRP1;, and URA3. A selectable marker for use in afilamentous fungal host cell may be selected from the group including,but not limited to, amdS (acetamidase), argB (ornithinecarbamoyltransferase), bar (phosphinothricin acetyltransferase), hygB(hygromycin phosphotransferase), niaD (nitrate reductase), pyrG(orotidine-5′-phosphate decarboxylase), sC (sulfate adenyltransferase),trpc (anthranilate synthase), and glufosinate resistance markers, aswell as equivalents from other species. Preferred for use in anAspergillus cell are the amdS and pyrG markers of Aspergillus nidulansor Aspergillus oryzae and the bar marker of Streptomyces hygroscopicus.Furthermore, selection may be accomplished by cotransformation, e.g., asdescribed in WO 91/17243, where the selectable marker is on a separatevector.

[0175] The procedures used to ligate the nucleic acid sequences codingfor the peptides and peptide conjugates, the promoter and theterminator, respectively, and to insert them into suitable vectorscontaining the information necessary for replication, are well known topersons skilled in the art (cf., for instance, Sambrook et al.,op.cit.).

[0176] The host cell into which the expression vector is introduced maybe any cell which is capable of producing the peptides and peptideconjugates and is may be a eukaryotic cell, such as invertebrate(insect) cells or vertebrate cells, e.g., Xenopus laevis oocytes ormammalian cells, in particular insect and mammalian cells. Examples ofsuitable mammalian cell lines are the COS (e.g., ATCC CRL 1650), BHK(e.g., ATCC CRL 1632, ATCC CCL 10) or CHO (e.g., ATCC CCL 61) celllines. Methods for transfecting mammalian cells and expressing DNAsequences introduced in the cells are described in e.g., Kaufman andSharp, 1982, J. Mol. Biol. 159:601-621; Southern and Berg, 1982, J. Mol.Appl. Genet. 1:327-341; Loyter et al., 1982, Proc. Natl. Acad. Sci. USA79:422-426; Wigler et al., 1978, Cell 14:725; Corsaro and Pearson, 1981,Somatic Cell Genetics 7:603, Graham and van der Eb, 1973, Virology52:456; Fraley et al., 1980, JBC 225:10431; Capecchi, 1980, Cell 22:479;Wiberg et al., 1983,NAR 11:7287; and Neumann et al., 1982, EMBO J.1:841-845. The host cell may also be a unicellular pathogen, e.g., aprokaryote, or a non-unicellular pathogen, e.g., a eukaryote. Usefulunicellular cells are bacterial cells such as gram positive bacteriaincluding, but not limited to, a Bacillus cell, e.g., Bacillusalkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacilluscirculans, Bacillus coagulans, Bacillus lautus, Bacillus lentus,Bacillus licheniformis, Bacillus megaterium, Bacillusstearothermophilus, Bacillus subtilis, and Bacillus thuringiensis; or aStreptomyces cell, e.g., Streptomyces lividans or Streptomyces murinus,or gram negative bacteria such as E. coli and Pseudomonas sp. In apreferred embodiment, the bacterial host cell is a Bacillus lentus,Bacillus licheniformis, Bacillus stearothermophilus or Bacillus subtiliscell. The transformation of a bacterial host cell may, for instance, beeffected by protoplast transformation (see, e.g., Chang and Cohen, 1979,Molecular General Genetics 168:111-115), by using competent cells (see,e.g., Young and Spizizin, 1961, Journal of Bacteriology 81:823-829, orDubnar and Davidoff Abelson, 1971, Journal of Molecular Biology56:209-221), by electroporation (see, e.g., Shigekawa and Dower, 1988,Biotechniques 6:742-751), or by conjugation (see, e.g., Koehler andThorne, 1987, Journal of Bacteriology 169:5771-5278). The host cell maybe a fungal cell. The fungal host cell may also be a yeast cell. “Yeast”as used herein includes ascosporogenous yeast (Endomycetales),basidiosporogenous yeast, and yeast belonging to the Fungi Imperfecti(Blastomycetes).

[0177] The medium used to culture the cells may be any conventionalmedium suitable for growing mammalian cells, such as a serum-containingor serum-free medium containing appropriate supplements, or a suitablemedium for growing insect, yeast or fungal cells. Suitable media areavailable from commercial suppliers or may be prepared according topublished recipes (e.g,. in catalogues of the American Type CultureCollection).

[0178] Thus, the invention also relates to a method for producing theexendin variants and peptide conjugates of the invention having anatural polypeptide sequence, comprising

[0179] a) introducing a nucleic acid sequence encoding a polypeptidesequence comprising the peptide sequence of the exendin variant or thepeptide conjugate of the invention and a selectable marker containedwithin a nucleic acid construct or a vector into a host cell to obtain arecombinant host cell;

[0180] b) selecting said recombinant host cell;

[0181] c) culturing said recombinant host cells under conditionspermitting the production of said polypeptide sequence;

[0182] d) isolating said polypeptide sequence from the culture; and

[0183] e) optionally cleaving said polypeptide sequence using anappropriate protease to obtain said peptide conjugate.

[0184] The variants and peptide conjugates of the invention having anatural polypeptide sequence thus produced by the cells may then berecovered from the culture medium by conventional procedures includingseparating the host cells from the medium by centrifugation orfiltration, precipitating the proteinaceous components of thesupernatant or filtrate by means of a salt, e.g., ammonium sulphate,purification by a variety of chromatographic procedures, e.g., ionexchange chromatography, affinity chromatography, or the like. Thelipophilic substituent(s) may be attached to the peptide of the presentinvention using procedures known in the art. In one embodiment, thelipophilic substituent may be attached by incorporating an amino acidwith the lipophilic substituent already attached in the standardsynthesis method (see, for example, synthesis of compound 7 in theExamples section). Alternatively, the substituent may be attached afterthe peptide has been synthesized and isolated as, for example, describedin WO98/08871.

[0185] The invention is further illustrated by the following examples.

EXAMPLES

[0186] Peptide Synthesis, General Procedures

[0187] Apparatus and Synthetic Strategy

[0188] Peptides are synthesized batchwise in a polyethylene vesselequipped with a polypropylene filter for filtration using9-fluorenylmethyloxycarbonyl (Fmoc) as the N-α-amino protecting groupand suitable common protection groups for side-chain functionalities(Dryland et al., 1986, J. Chem. Soc., Perkin Trans. 1:125-137).

[0189] Solvents

[0190] Solvent DMF (N,N-dimethylformamide, Riedel de-Häen, Germany) ispurified by passing it through a column packed with a strong cationexchange resin (Lewatit S 100 MB/H strong acid, Bayer AG Leverkusen,Germany) and analysed for free amines prior to use by addition of3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine (Dhbt-OH) giving rise toa yellow color (Dhbt-O-anion) if free amines are present. Solvent DCM(dichloromethane, analytical grade, Riedel de-Häen, Germany) is useddirectly without purification. THF (tetrahydrofuran, analytical grade,Riedel de-Haen, Germany) is used directly without further purification.

[0191] Amino Acids

[0192] Fmoc-protected amino acids are purchased from MilliGen (UK) andfrom PerSeptive Biosystems GmbH Hamburg, Germany in suitable side-chainprotected forms. FmocLys(palmitoyl)-OH is purchased from Bachem(Switzerland).

[0193] Linker

[0194] (4-hydroxymethylphenoxy)acetic acid (HMPA), Novabiochem,Switzerland is coupled to the resin either as a preformed or in situgenerated 1-hydroxybenzotriazole (HObt) ester by means of DIC.

[0195] Coupling Reagents

[0196] Coupling reagent diisopropylcarbodiimide (DIC) is purchased from(Riedel de-,Häen, Germany) and distilled prior to use,dicyclohexylcarbodiimide (DCC) is purchased from Merck-Schuchardt,München, Germany, and purified by distillation.

[0197] Solid Supports

[0198] Peptides synthesized according to the Fmoc-strategy aresynthesized on the following types of solid support using 0.05 M orhigher concentrations of Fmoc-protected activated amino acid in DMF.TentaGel S resins 0.22-0.31 mmol/g (TentaGel S-Ram, TentaGel SRAM-Lys(Boc)Fmoc; Rapp polymere, Germany).

[0199] Catalysts and other Reagents

[0200] Diisopropylethylamine (DIEA) is purchased from Aldrich, Germany,and ethylenediamine from Fluka, piperidine and pyridine from Riedel-deHäen, Frankfurt, Germany. 4-(N,N-di-methylamino)pyridine (DMAP) ispurchased from Fluka, Switzerland and used as a catalyst in couplingreactions involving symmetrical anhydrides. Ethanedithiol is purchasedfrom Riedel-de Häen, Frankfurt, Germany.3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine (Dhbt-OH) and1-hydroxybenzotriazole (HObt) are obtained from Fluka, Switzerland.

[0201] Coupling Procedures

[0202] The first amino acid is coupled as a symmetrical anhydride in DMFgenerated from the appropriate N-α-protected amino acid by means of DICor DCC. The following amino acids are coupled as preformed HObt estersmade from appropriate N-α-protected amino acids and HObt by means of DICin DMF. Acylations are checked by the ninhydrin test performed at 80° C.in order to prevent Fmoc deprotection during the test (Larsen, B. D. andHolm, A., 1994, Int. J. Peptide Protein Res. 43:1-9).

[0203] Coupling as HObt-ester

[0204] Method a. 3 eq. N-α-amino protected amino acid is dissolved inDMF together with 3 eq. HObt and 3 eq DIC. The solution is left at r.t.for 10 minutes and then added to the resin, which had been washed with asolution of 0.2% Dhbt-OH in DMF prior to the addition of thepreactivated amino acid.

[0205] Method b. 3 eq. N-α-amino protected amino acid is dissolved inDMF together with 3 eq. HObt. 3 eq DIC are added just prior to use. Thefinal solution is added to the resin.

[0206] Preformed Symmetrical Anhydride

[0207] 6 eq. N-α-amino protected amino acid is dissolved in DCM andcooled to 0° C. DCC or DIC (3 eq.) is added and the reaction continuedfor 10 min. The solvent is removed in vacuo and the residue dissolved inDMF. The DMF-solution is filtered in case of using DCC and immediatelyadded to the resin followed by 0.1 eq. of DMAP.

[0208] Deprotection of the N-α-amino Fmoc Protecting Group

[0209] Deprotection of the Fmoc group is performed by treatment with 20%piperidine in DMF (1×5 and 1×10 min.), followed by wash with DMF untilno yellow colour (Dhbt-O—) could be detected after addition of Dhbt-OHto the drained DMF.

[0210] Cleavage of Peptide from Resin with Acid

[0211] Method a Peptides are cleaved from the resins by treatment with95% trifluoroacetic acid (TFA, Riedel-de Häen, Frankfurt, Germany)-waterv/v or with 95% TFA and 5% ethanedithiol v/v at r.t. for 2 h. Thefiltered resins are washed with 95% TFA-water and filtrates and washingsare diluted by adding 10% acetic acid. The resulting mixture isextracted 3 times with ether and finally freeze dried. The crude freezedried product is analysed by high-performance liquid chromatography(HPLC) and identified by mass spectrometry (MS).

[0212] Batchwise Peptide Synthesis on TentaGel S-RAM

[0213] TentaGel S-RAM resin (100-1000 mg, 0.22-0.31 mmol/g) is placed ina polyethylene vessel equipped with a polypropylene filter forfiltration. The resin is swelled in DMF (5-10 ml), and the Fmoc group isremoved according to the procedure described above. The following aminoacids according to the sequence are coupled as Fmoc-protected HObtesters (3 eq.) generated in situ by means of DIC as described above. Thecouplings are continued for 3 h, unless otherwise specified. The resinis drained and washed with DMF (4×5−10 ml, 2 min each) in order toremove excess reagent. All acylations are checked by the ninhydrin testperformed at 80° C. After completion of the synthesis, the peptide-resinis washed with DMF (3×5−10 ml, 5 min each), DCM (3×5−10 ml, 1 min each)and finally diethyl ether (3×5−10 ml, 1 min each) and dried in vacuo.

[0214] HPLC Conditions

[0215] Isocratic HPLC analysis is preformed on a Shimadzu systemconsisting of an LC-6A pump, an MERCK HITACHI L-4000 UV detectoroperated at 215 nm and a Rheodyne 7125 injection valve with a 20 μlloop. The column used for isocratic analysis is a Spherisorb ODS-2(100×3 mm; 5-μm particles) (MicroLab, Aarhus, Denmark). HPLC analysisusing gradients is performed on a MERCK-HITACHI L-6200 Intelligent pump,an MERCK HITACHI L-4000 UV detector operated at 215 nm and a Rheodyne7125 injection valve with a 20 μl loop, or on a Waters 600 E instrumentequipped with a Waters 996 photodiode array detector. The columns usedare a Rescorce™ RPC 1 ml (Waters) or a LiChroCART 125-4, LiChrospher 100RP-18 (5 μm) (Merck).

[0216] Buffer A is 0.1 vol % TFA in water and buffer B 90 vol %acetonitrile, 9.9 vol % water and 0.1 vol % TFA. The buffers are pumpedthrough the columns at a flow rate of 1.3-1.5 ml/min using either of thefollowing gradients for peptide analysis 1) Linear gradient from 0%-100%B (30 min) or 2) 0% B (2 min) linear gradient from 0-50% B (23 min)50-100% B (5 min).

[0217] For Preparative HPLC, purification is performed on a Waters 600 Einstrument equipped with a Waters 996 photodiode array detector. Thecolumn used is a Waters Delta-Pak C-18 15 μm, 100 Å, 25×100 mm. Gradient“2)” is used with a flow rate of 9 ml/min.

[0218] Mass Spectroscopy

[0219] Mass spectra are obtained on a Finnigan Mat LCQ instrumentequipped with an electrospray (ESI) probe (ES-MS) and on a TofSpec E,Fisons Instrument (MALDI-TOF) using β-cyano-p-hydroxycinnamic acid asmatrix. Alternatively, spectra may be obtained by a Micromass LCTinstrument.

[0220] Peptide Synthesis of Prior Art Peptides

[0221] (i) Peptide synthesis of Compound (i),H-His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH₂

[0222] (exendin-4(1-39)-NH₂) (SEQ ID NO:102) on TentaGel S-RAM.

[0223] Dry TentaGel S-RAM resin (0.25 mmol/g, 1000 mg) is placed in apolyethylene vessel equipped with a polypropylene filter for filtrationand swelled for two hours in DMF (5 ml). The Fmoc group is removedaccording to the procedure described above, and the peptide according tothe sequence is assembled as described under “Batchwise peptidesynthesis on TentaGel S-RAM resins”. After completion of the synthesis,the peptide-resin is washed with DMF (3×5 ml, 1 min each), DCM (3×5 ml,1 min each), diethyl ether (3×5 ml, 1 min each) and dried in vacuo. Thepeptide is cleaved from the resin according to method a as describedabove and freeze dried from acetic acid. The crude peptide is purifiedby preparative HPLC using the procedure described above. The purifiedproduct is found to be homogeneous and the purity is found to be betterthan 90%. The identity of the peptide is confirmed by ES-MS. Yield 17%.

[0224] (ii) Peptide synthesis of Compound (ii),H-His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-NH₂(SEQID NO: 129)

[0225] (des Ser³⁹ exendin-4(1-39)-NH₂) (SEQ ID NO: 129) on TentaGelS-RAM.

[0226] Dry TentaGel S-RAM resin (0.25 mmol/g, 1000 mg) is placed in apolyethylene vessel equipped with a polypropylene filter for filtrationand swelled for two hours in DMF (5 ml). The Fmoc group is removedaccording to the procedure described above, and the peptide according tothe sequence is assembled as described under “Batchwise peptidesynthesis on TentaGel S-RAM resins”. After completion of the synthesis,the peptide-resin is washed with DMF (3×5 ml, 1 min each), DCM (3×5 ml,1 min each), diethyl ether (3×5 ml, 1 min each) and dried in vacuo. Thepeptide is cleaved from the resin according to method a as describedabove and freeze dried from acetic acid. The crude peptide is purifiedby preparative HPLC using the procedure described above. The purifiedproduct is found to be homogeneous and the purity is found to be betterthan 97%. The identity of the peptide is confirmed by ES-MS. Yield 22%.

[0227] (iii) Peptide synthesis of Compound (iii),H-His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-NH₂(SEQ ID NO: 87)

[0228] (Gly⁸-GLP1-(7-36)(Human)-NH₂) (SEQ ID NO:87) on TentaGel S-RAM.

[0229] Dry TentaGel S-RAM resin (0.25 mmol/g, 1000 mg) is placed in apolyethylene vessel equipped with a polypropylene filter for filtrationand swelled for two hours in DMF (5 ml). The Fmoc group is removedaccording to the procedure described above, and the peptide according tothe sequence is assembled as described under “Batchwise peptidesynthesis on TentaGel S-RAM resins”. After completion of the synthesis,the peptide-resin is washed with DMF (3×5 ml, 1 min each), DCM (3×5 ml,1 min each), diethyl ether (3×5 ml, 1 min each) and dried in vacuo. Thepeptide is cleaved from the resin according to method a as describedabove and freeze dried from acetic acid. The crude peptide is purifiedby preparative HPLC using the procedure described above. The purifiedproduct is found to be homogeneous and the purity is found to be betterthan 95%. The identity of the peptide is confirmed by ES-MS. Yield 9%.

[0230] Synthesis of Peptide Sequences of the Invention

[0231] 1. Peptide synthesis of Compound 1,H-His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Ser-NH₂(SEQ ID NO: 101)

[0232] (des Pro³⁶-exendin-4(1-39)-NH₂) (SEQ ID NO:101) on TentaGelS-RAM.

[0233] Dry TentaGel S-RAM resin (0.25 mmol/g, 1500 mg) is placed in apolyethylene vessel equipped with a polypropylene filter for filtrationand swelled for two hours in DMF (5 ml). The Fmoc group is removedaccording to the procedure described above, and the peptide according tothe sequence is assembled as described under “Batchwise peptidesynthesis on TentaGel S-RAM resins”. After completion of the synthesis,the peptide-resin is washed with DMF (3×5 ml, 1 min each), DCM (3×5 ml,1 min each), diethyl ether (3×5 ml, 1 min each) and dried in vacuo. Thepeptide is cleaved from the resin according to method a as describedabove and freeze dried from acetic acid. The crude peptide is purifiedby preparative HPLC using the procedure described above. The purifiedproduct is found to be homogeneous and the purity is found to be betterthan 95%. The identity of the peptide is confirmed by ES-MS. Yield18.3%.

[0234] 2. Peptide synthesis of Compound 2,H-His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Ser-(LYS)₆-NH₂(SEQ ID NO: 93)

[0235] (des-Pro³⁶-exendin-4(1-39)-Lys₆-NH₂) (SEQ ID NO:93) on TentaGelS-RAM-Lys(Boc)Fmoc.

[0236] Dry TentaGel S-RAM-Lys(Boc)Fmoc resin (0.22 mmol/g, 1500 mg) isplaced in a poly-ethylene vessel equipped with a polypropylene filterfor filtration and swelled for two hours in DMF (5 ml). The Fmoc groupon the first lysine is removed as described above and the synthesis iscontinued until finishing the peptide sequence as described under“Batchwise peptide synthesis on TentaGel S-Ram-Lys(Boc)Fmoc”. Aftercompletion of the synthesis, the peptide-resin is washed with DMF (3×5ml, 1 min each), DCM (3×5 ml, 1 min each), diethyl ether (3×5 ml, 1 mineach) and dried in vacuo. The peptide is cleaved from the resinaccording to method a as described above and freeze dried from aceticacid. The crude freeze dried product is purified by preparative HPLCusing the procedure described above. The purified product is found to behomogeneous and the purity is found to be better than 95%. The identityof the peptide is confirmed by ES-MS. Yield 22.1%.

[0237] 3. Peptide synthesis of Compound 3,H-His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-(Lys)₆-NH₂(SEQ ID NO: 92)

[0238] (exendin-4(1-39)-Lys₆-NH₂) (SEQ ID NO:92) on TentaGelS-RAM-Lys(Boc)Fmoc.

[0239] Dry TentaGel S-RAM-Lys(Boc)Fmoc resin (0.22 mmol/g, 1000 mg) isplaced in a poly-ethylene vessel equipped with a polypropylene filterfor filtration and swelled for two hours in DMF (5 ml). The Fmoc groupon the first lysine is removed as described above and the synthesis iscontinued until finishing the peptide sequence as described under“Batchwise peptide synthesis on TentaGel S-Ram-Lys(Boc)Fmoc”. Aftercompletion of the synthesis, the peptide-resin is washed with DMF (3×5ml, 1 min each), DCM (3×5 ml, 1 min each), diethyl ether (3×5 ml, 1 mineach) and dried in vacuo. The peptide is cleaved from the resinaccording to method a as described above and freeze dried from aceticacid. The crude freeze dried product is purified by preparative HPLCusing the procedure described above. The purified product is found to behomogeneous and the purity is found to be better than 90%. The identityof the peptide is confirmed by ES-MS. Yield 20.5%.

[0240] 4. Peptide synthesis of Compound 4,H-His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-(Lys)₆-NH₂(SEQ ID NO: 88) (Gly⁸-GLP1-(7-36)(Human)-Lys₆-NH₂) (SEQ ID NO:88) onTentaGel S-RAM-Lys(Boc)Fmoc.

[0241] Dry TentaGel S-RAM-Lys(Boc)Fmoc resin (0.22 mmol/g, 1000 mg) isplaced in a polyethylene vessel equipped with a polypropylene filter forfiltration and swelled for two hours in DMF (5 ml). The Fmoc group onthe first lysine is removed as described above and the synthesis iscontinued until finishing the peptide sequence as described under“Batchwise peptide synthesis on TentaGel S-Ram-Lys(Boc)Fmoc”. Aftercompletion of the synthesis, the peptide-resin is washed with DMF (3×5ml, 1 min each), DCM (3×5 ml, 1 min each), diethyl ether (3×5 ml, 1 mineach) and dried in vacuo. The peptide is cleaved from the resinaccording to method a as described above and freeze dried from aceticacid. The crude freeze dried product is purified by preparative HPLCusing the procedure described above. The purified product is found to behomogeneous and the purity is found to be better than 95%. The identityof the peptide is confirmed by ES-MS. Yield 11.7%.

[0242] 4a. Peptide synthesis of Compound 4,H-His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-Lys-Lys-Lys-Lys-Lys-Lys-NH₂(SEQ ID NO: 88)

[0243] ([Gly⁸]hGLP-1(7-36)-(Lys)₆-NH₂) (SEQ ID NO:88) on TentaGelS-RAM-Lys(Boc)Fmoc.

[0244] Dry TentaGel S-RAM-Lys(Boc)Fmoc resin (0.22 mmol/g, 2013 mg) isplaced in a glass vessel equipped with a polypropylene filter forfiltration and swelled for two hours in DMF (5 ml). The Fmoc group onthe first lysine is removed as described above and the synthesis iscontinued until finishing the peptide sequence as described under“Batchwise peptide synthesis on TentaGel S-Ram-Lys(Boc)Fmoc”. Aftercompletion of the synthesis, the peptide-resin is washed with DMF (3×5ml, 1 min each), DCM (3×5 ml, 1 min each), diethyl ether (3×5 ml, 1 mineach) and dried in vacuo. The peptide is cleaved from the resinaccording to method a as described above and freeze dried from aceticacid. The crude freeze dried product is purified by preparative HPLCusing the procedure described above. The purified product is found to behomogeneous and the purity is found to be better than 90%. The identityof the peptide is confirmed by ES-MS. Yield 13%.

[0245] 5. Peptide synthesis of Compound 5,H-His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-Lys(palmitoyl)-(Lys)₆-NH₂(SEQ ID NO: 89)

[0246] ([Gly⁸, Lys³⁷(palmitoyl)]GLP1-(7-36)(Human)-(Lys)₇-NH₂) (SEQ IDNO:89) on TentaGel S-RAM-Lys(Boc)Fmoc.

[0247] Dry TentaGel S-RAM-Lys(Boc)Fmoc resin (0.22 mmol/g, 1000 mg) isplaced in a polyethylene vessel equipped with a polypropylene filter forfiltration and swelled for two hours in DMF (5 ml). The Fmoc group onthe first lysine is removed as described above and the synthesis iscontinued until finishing the peptide sequence as described under“Batchwise peptide synthesis on TentaGel S-Ram-Lys(Boc)Fmoc”. Thereagent Fmoc-Lys(palmitoyl)-OH is coupled in a slightly modified mannerdue to its poor solubility in DMF. Approximately 400 mg ofFmoc-Lys(palmitoyl)-OH is dissolved in approximately 6 ml THF ratherthan DMF. After completion of the synthesis, the peptide-resin is washedwith DMF (3×5 ml, 1 min each), DCM (3×5 ml, 1 min each), diethyl ether(3×5 ml, 1 min each) and dried in vacuo. The peptide is cleaved from theresin according to method b as described above and freeze dried fromacetic acid. The crude freeze dried product is purified by preparativeHPLC using the procedure described above. The purified product is foundto be homogeneous and the purity is found to be better than 95%. Theidentity of the peptide is confirmed by ES-MS. Yield 9.3%.

[0248] 6. Peptide synthesis of Compound 6,H-His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys(palmitoyl)-Gly-Arg-(Lys)₆-NH₂(SEQ ID NO: 90)

[0249] ([Gly⁸ Lys³⁴(palmitoyl)]GLP1-(7-36)(Human)-(Lys)₆-NH₂) (SEQ IDNO:90) on TentaGel S-RAM-Lys(Boc)Fmoc.

[0250] Dry TentaGel S-RAM-Lys(Boc)Fmoc resin (0.22 mmol/g, 1000 mg) isplaced in a polyethylene vessel equipped with a polypropylene filter forfiltration and swelled for two hours in DMF (5 ml). The Fmoc group onthe first lysine is removed as described above and the synthesis iscontinued until finishing the peptide sequence as described under“Batchwise peptide synthesis on TentaGel S-Ram-Lys(Boc)Fmoc”. Thereagent Fmoc-Lys(palmitoyl)-OH is coupled in a slightly modified mannerdue to its poor solubility in DMF. Approximately 400 mg ofFmoc-Lys(palmitoyl)-OH is dissolved in approximately 6 ml THF ratherthan DMF. After completion of the synthesis, the peptide-resin is washedwith DMF (3×5 ml, 1 min each), DCM (3×5 ml, 1 min each), diethyl ether(3×5 ml, 1 min each) and dried in vacuo. The peptide is cleaved from theresin according to method a as described above and freeze dried fromacetic acid. The crude freeze dried product is purified by preparativeHPLC using the procedure described above. The purified product is foundto be homogeneous and the purity is found to be better than 90%. Theidentity of the peptide is confirmed by ES-MS. Yield 4.2%.

[0251] 7. Peptide synthesis of Compound 7,H-His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys(palmitoyl)-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-(Lys)₆-NH₂(SEQ ID NO: 103)

[0252] ([Gly⁸, Lys²⁶(palmitoyl)]GLP1-(7-36)(Human)-(Lys)₆-NH₂) (SEQ IDNO:103) on TentaGel S-RAM-Lys(Boc)Fmoc.

[0253] Dry TentaGel S-RAM-Lys(Boc)Fmoc resin (0.22 mmol/g, 1000 mg) isplaced in a polyethylene vessel equipped with a polypropylene filter forfiltration and swelled for two hours in DMF (5 ml). The Fmoc group onthe first lysine is removed as described above and the synthesis iscontinued until finishing the peptide sequence as described under“Batchwise peptide synthesis on TentaGel S-Ram-Lys(Boc)Fmoc”. Thereagent Fmoc-Lys(palmitoyl)-OH is coupled in a slightly modified mannerdue to its poor solubility in DMF. Approximately 400 mg ofFmoc-Lys(palmitoyl)-OH is dissolved in approximately 6 ml THF ratherthan DMF. After completion of the synthesis, the peptide-resin is washedwith DMF (3×5 ml, 1 min each), DCM (3×5 ml, 1 min each), diethyl ether(3×5 ml, 1 min each) and dried in vacuo. The peptide is cleaved from theresin according to method a as described above and freeze dried fromacetic acid. The crude freeze dried product is purified by preparativeHPLC using the procedure described above. The purified product is foundto be homogeneous and the purity is found to be better than 90%. Theidentity of the peptide is confirmed by ES-MS. Yield 2.2%.

[0254] 8. Peptide synthesis of Compound 8,H-Lys-Lys-Lys-Lys-Lys-Lys-His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Ser-NH₂(SEQ ID NO: 149)

[0255] (H-(Lys)₆-des Pro³⁶exendin-4(1-39)-NH₂) (SEQ ID NO: 149) onTentaGel S-RAM-Fmoc.

[0256] Dry TentaGel S-RAM-Fmoc resin (0.23 mmol/g, 1000 mg) is placed ina poly-ethylene vessel equipped with a polypropylene filter forfiltration and swelled for two hours in DMF (5 ml). The Fmoc group onthe resin is removed as described above and the synthesis is continueduntil finishing the peptide sequence as described under “Batchwisepeptide synthesis on TentaGel S-Ram-Fmoc”. After completion of thesynthesis, the peptide-resin is washed with DMF (3×5 ml, 1 min each),DCM (3×5 ml, 1 min each), diethyl ether (3×5 ml, 1 min each) and driedin vacuo. The peptide is cleaved from the resin according to method a asdescribed above and freeze dried from acetic acid. The crude freezedried product is purified by preparative HPLC using the proceduredescribed above. The purified product is found to be homogeneous and thepurity is found to be better than 95%. The identity of the peptide isconfirmed by ES-MS. Yield 26%.

[0257] 9. Peptide synthesis of Compound 9,H-Lys₆-His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Ser-(Lys)₆-NH₂(SEQ ID NO: 150)

[0258] (H-Lys₆-des Pro³⁶exendin-4(1-39)-Lys₆-NH₂) (SEQ ID NO: 150) onTentaGel S-RAM-Lys(Boc)Fmoc.

[0259] Dry TentaGel S-RAM-Lys(Boc)Fmoc resin (0.22 mmol/g, 1000 mg) isplaced in a poly-ethylene vessel equipped with a polypropylene filterfor filtration and swelled for two hours in DMF (5 ml). The Fmoc groupon the first lysine is removed as described above and the synthesis iscontinued until finishing the peptide sequence as described under“Batchwise peptide synthesis on TentaGel S-Ram-Lys(Boc)Fmoc”. Aftercompletion of the synthesis, the peptide-resin is washed with DMF (3×5ml, 1 min each), DCM (3×5 ml, 1 min each), diethyl ether (3×5 ml, 1 mineach) and dried in vacuo. The peptide is cleaved from the resinaccording to method a as described above and freeze dried from aceticacid. The crude freeze dried product is purified by preparative HPLCusing the procedure described above. The purified product is found to behomogeneous and the purity is found to be better than 90%. The identityof the peptide is confirmed by ES-MS. Yield 32%.

[0260] 10. Peptide synthesis of Compound 10,H-Lys-Lys-Lys-Lys-Lys-Lys-His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-Lys-Lys-Lys-Lys-Lys-Lys-NH₂(SEQ ID NO: 118) (H-(Lys)₆-([Gly⁸]hGLP-1(7-36)-(Lys)₆-NH₂) (SEQ ID NO:118) on TentaGel S-RAM-Lys(Boc)Fmoc.

[0261] Dry TentaGel S-RAM-Lys(Boc)Fmoc resin (0.22 mmol/g, 1000 mg) isplaced in a poly-ethylene vessel equipped with a polypropylene filterfor filtration and swelled for two hours in DMF (5 ml). The Fmoc groupon the first lysine is removed as described above and the synthesis iscontinued until finishing the peptide sequence as described under“Batchwise peptide synthesis on TentaGel S-Ram-Lys(Boc)Fmoc”. Aftercompletion of the synthesis, the peptide-resin is washed with DMF (3×5ml, 1 min each), DCM (3×5 ml, 1 min each), diethyl ether (3×5 ml, 1 mineach) and dried in vacuo. The peptide is cleaved from the resinaccording to method a as described above and freeze dried from aceticacid. The crude freeze dried product is purified by preparative HPLCusing the procedure described above. The purified product is found to behomogeneous and the purity is found to be better than 90%. The identityof the peptide is confirmed by ES-MS. Yield 18%.

[0262] 11. Peptide synthesis of Compound 11,H-Lys-Lys-Lys-Lys-Lys-Lys-His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-NH₂(SEQ ID NO: 119)

[0263] (H-(Lys)₆-[Gly⁸]hGLP-1(7-36)-NH₂) (SEQ ID NO: 119) on TentaGelS-RAM-Lys(Boc)Fmoc.

[0264] Dry TentaGel S-RAM-Fmoc resin (0.23 mmol/g, 1000 mg) is placed ina poly-ethylene vessel equipped with a polypropylene filter forfiltration and swelled for two hours in DMF (5 ml). The Fmoc group onthe resin is removed as described above and the synthesis is continueduntil finishing the peptide sequence as described under “Batchwisepeptide synthesis on TentaGel S-Ram-Fmoc”. After completion of thesynthesis, the peptide-resin is washed with DMF (3×5 ml, 1 min each),DCM (3×5 ml, 1 min each), diethyl ether (3×5 ml, 1 min each) and driedin vacuo. The peptide is cleaved from the resin according to method a asdescribed above and freeze dried from acetic acid. The crude freezedried product is purified by preparative HPLC using the proceduredescribed above. The purified product is found to be homogeneous and thepurity is found to be better than 98%. The identity of the peptide isconfirmed by ES-MS. Yield 15%.

[0265] 12. Peptide synthesis of Compound 12,H-His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-Lys-Lys-Lys-Lys-Lys-Lys-Lys-Lys-NH₂(SEQ ID NO: 120)

[0266] ([Gly⁸]hGLP-1(7-36)-(Lys)₈-NH₂) (SEQ ID NO: 120) on TentaGelS-RAM-Lys(Boc)Fmoc.

[0267] Dry TentaGel S-RAM-Lys(Boc)Fmoc resin (0.22 mmol/g, 1000 mg) isplaced in a poly-ethylene vessel equipped with a polypropylene filterfor filtration and swelled for two hours in DMF (5 ml). The Fmoc groupon the first lysine is removed as described above and the synthesis iscontinued until finishing the peptide sequence as described under“Batchwise peptide synthesis on TentaGel S-Ram-Lys(Boc)Fmoc”. Aftercompletion of the synthesis, the peptide-resin is washed with DMF (3×5ml, 1 min each), DCM (3×5 ml, 1 min each), diethyl ether (3×5 ml, 1 mineach) and dried in vacuo. The peptide is cleaved from the resinaccording to method a as described above and freeze dried from aceticacid. The crude freeze dried product is purified by preparative HPLCusing the procedure described above. The purified product is found to behomogeneous and the purity is found to be better than 98%. The identityof the peptide is confirmed by ES-MS. Yield 4,2%.

[0268] 13. Peptide synthesis of Compound 13,H-His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-Lys-Lys-Lys-Lys-Lys-Lys-Lys-Lys-Lys-Lys-NH₂(SEQ ID NO: 121)

[0269] ([Gly⁸]hGLP-1(7-36)-(Lys)₁₀-NH₂) (SEQ ID NO: 121) on TentaGelS-RAM-Lys(Boc)Fmoc.

[0270] Dry TentaGel S-RAM-Lys(Boc)Fmoc resin (0.22 mmol/g, 1000 mg) isplaced in a poly-ethylene vessel equipped with a polypropylene filterfor filtration and swelled for two hours in DMF (5 ml). The Fmoc groupon the first lysine is removed as described above and the synthesis iscontinued until finishing the peptide sequence as described under“Batchwise peptide synthesis on TentaGel S-Ram-Lys(Boc)Fmoc”. Aftercompletion of the synthesis, the peptide-resin is washed with DMF (3×5ml, 1 min each), DCM (3×5 ml, 1 min each), diethyl ether (3×5 ml, 1 mineach) and dried in vacuo. The peptide is cleaved from the resinaccording to method a as described above and freeze dried from aceticacid. The crude freeze dried product is purified by preparative HPLCusing the procedure described above. The purified product is found to behomogeneous and the purity is found to be better than 95%. The identityof the peptide is confirmed by ES-MS. Yield 2%.

[0271] 14. Peptide synthesis of Compound 14,H-His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gin-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Ser-NH₂(SEQ ID NO: 132)

[0272] (H-des Pro³⁶, Pro³⁷, Pro³⁸exendin-4(1-39)-NH₂) (SEQ ID NO: 132)on TentaGel S-RAM-Fmoc.

[0273] Dry TentaGel S-RAM-Fmoc resin (0.23 mmol/g, 1000 mg) is placed ina poly-ethylene vessel equipped with a polypropylene filter forfiltration and swelled for two hours in DMF (5 ml). The Fmoc group onthe resin is removed as described above and the synthesis is continueduntil finishing the peptide sequence as described under “Batchwisepeptide synthesis on TentaGel S-Ram-Fmoc”. After completion of thesynthesis, the peptide-resin is washed with DMF (3×5 ml, 1 min each),DCM (3×5 ml, 1 min each), diethyl ether (3×5 ml, 1 min each) and driedin vacuo. The peptide is cleaved from the resin according to method a asdescribed above and freeze dried from acetic acid. The crude freezedried product is purified by preparative HPLC using the proceduredescribed above. The purified product is found to be homogeneous and thepurity is found to be better than 95%. The identity of the peptide isconfirmed by ES-MS. Yield 11%.

[0274] 15. Peptide synthesis of Compound 15,H-Lys-Lys-Lys-Lys-Lys-Lys-His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Ser-NH₂(SEQ ID NO: 134)

[0275] (H-(Lys)₆-des Pro³⁶, Pro³⁷, Pro³⁸-exendin-4(1-39)-NH₂) (SEQ IDNO: 134) on TentaGel S-RAM-Fmoc.

[0276] Dry TentaGel S-RAM-Fmoc resin (0.23 mmol/g, 1000 mg) is placed ina poly-ethylene vessel equipped with a polypropylene filter forfiltration and swelled for two hours in DMF (5 ml). The Fmoc group onthe resin is removed as described above and the synthesis is continueduntil finishing the peptide sequence as described under “Batchwisepeptide synthesis on TentaGel S-Ram-Fmoc”. After completion of thesynthesis, the peptide-resin is washed with DMF (3×5 ml, 1 min each),DCM (3×5 ml, 1 min each), diethyl ether (3×5 ml, 1 min each) and driedin vacuo. The peptide is cleaved from the resin according to method a asdescribed above and freeze dried from acetic acid. The crude freezedried product is purified by preparative HPLC using the proceduredescribed above. The purified product is found to be homogeneous and thepurity is found to be better than 94%. The identity of the peptide isconfirmed by ES-MS. Yield 17%.

[0277] 16. Peptide synthesis of compound 16,H-Asn-Glu-Glu-Glu-Glu-Glu-His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-le-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Ser-NH₂(SEQ ID NO: 137)

[0278] (H-Asn-(Glu)-des Pro, Pro³, Pro³⁸exendin-4(1-39)-NH²) (SEQ ID NO:137) on TentaGel S-RAM-Fmoc.

[0279] Dry TentaGel S-RAM-Fmoc resin (0.23 mmol/g, 1000 mg) is placed ina poly-ethylene vessel equipped with a polypropylene filter forfiltration and swelled for two hours in DMF (5 ml). The Fmoc group onthe resin is removed as described above and the synthesis is continueduntil finishing the peptide sequence as described under “Batchwisepeptide synthesis on TentaGel S-Ram-Fmoc”. After completion of thesynthesis, the peptide-resin is washed with DMF (3×5 ml, 1 min each),DCM (3×5 ml, 1 min each), diethyl ether (3×5 ml, 1 min each) and driedin vacuo. The peptide is cleaved from the resin according to method a asdescribed above and freeze dried from acetic acid. The crude freezedried product is purified by preparative HPLC using the proceduredescribed above. The purified product is found to be homogeneous and thepurity is found to be better than 90%. The identity of the peptide isconfirmed by ES-MS. Yield 9%.

[0280] 17. Peptide synthesis of Compound 17, Compound 3,H-(Lys)₆-His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Ser-(Lys)₆-NH₂(SEQ ID NO: 135)

[0281] (H-(Lys)₆-des Pro³⁶, Pro³⁷, Pro³⁸exendin-4(1-39)-(Lys)₆-NH₂) (SEQID NO: 135) on TentaGel S-RAM-Lys(Boc)Fmoc.

[0282] Dry TentaGel S-RAM-Lys(Boc)Fmoc resin (0.22 mmol/g, 1000 mg) isplaced in a poly-ethylene vessel equipped with a polypropylene filterfor filtration and swelled for two hours in DMF (5 ml), The Fmoc groupon the first lysine is removed as described above and the synthesis iscontinued until finishing the peptide sequence as described under“Batchwise peptide synthesis on TentaGel S-Ram-Lys(Boc)Fmoc”. Aftercompletion of the synthesis, the peptide-resin is washed with DMF (3×5ml, 1 min each), DCM (3×5 ml, 1 min each), diethyl ether (3×5 ml, 1 mineach) and dried in vacuo. The peptide is cleaved from the resinaccording to method a as described above and freeze dried from aceticacid. The crude freeze dried product is purified by preparative HPLCusing the procedure described above. The purified product is found to behomogeneous and the purity is found to be better than 90%. The identityof the peptide is confirmed by ES-MS. Yield 10%.

[0283] 18. Peptide synthesis of Compound 18,H-Asn-Glu-Glu-Glu-Glu-Glu-His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Ser-(Lys)₆-NH₂(SEQ ID NO: 136)

[0284] (H-Asn-(Glu)₅-des Pro³⁶, Pro³⁷, Pro³⁸exendin-4(1-39)-(Lys)₆-NH₂)(SEQ ID NO: 136) on TentaGel S-RAM-Lys(Boc)Fmoc.

[0285] Dry TentaGel S-RAM-Lys(Boc)Fmoc resin (0.22 mmol/g, 1000 mg) isplaced in a poly-ethylene vessel equipped with a polypropylene filterfor filtration and swelled for two hours in DMF (5 ml). The Fmoc groupon the first lysine is removed as described above and the synthesis iscontinued until finishing the peptide sequence as described under“Batchwise peptide synthesis on TentaGel S-Ram-Lys(Boc)Fmoc”. Aftercompletion of the synthesis, the peptide-resin is washed with DMF (3×5ml, 1 min each), DCM (3×5 ml, 1 min each), diethyl ether (3×5 ml, 1 mineach) and dried in vacuo. The peptide is cleaved from the resinaccording to method a as described above and freeze dried from aceticacid. The crude freeze dried product is purified by preparative HPLCusing the procedure described above. The purified product is found to behomogeneous and the purity is found to be better than 92%. The identityof the peptide is confirmed by ES-MS. Yield 14%.

[0286] 19. Peptide synthesis of Compound 19,H-His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Ser-(Lys)₆-NH₂(SEQ ID NO: 133)

[0287] (des Pro³⁶, Pro³⁷, Pro³⁸exendin-4(1-39)-(Lys)₆-NH₂) (SEQ ID NO:133) on TentaGel S-RAM-Lys(Boc)Fmoc.

[0288] Dry TentaGel S-RAM-Lys(Boc)Fmoc resin (0.22 mmol/g, 1000 mg) isplaced in a poly-ethylene vessel equipped with a polypropylene filterfor filtration and swelled for two hours in DMF (5 ml). The Fmoc groupon the first lysine is removed as described above and the synthesis iscontinued until finishing the peptide sequence as described under“Batchwise peptide synthesis on TentaGel S-Ram-Lys(Boc)Fmoc”. Aftercompletion of the synthesis, the peptide-resin is washed with DMF (3×5ml, 1 min each), DCM (3×5 ml, 1 min each), diethyl ether (3×5 ml, 1 mineach) and dried in vacuo. The peptide is cleaved from the resinaccording to method a as described above and freeze dried from aceticacid. The crude freeze dried product is purified by preparative HPLCusing the procedure described above. The purified product is found to behomogeneous and the purity is found to be better than 97%. The identityof the peptide is confirmed by ES-MS. Yield 19%.

[0289] 20. Recombinant Preparation of Compound 2

[0290] Construction of the pYES0010 Expression Vector

[0291] A synthetic cDNA was constructed for heterolog expression inyeast. The protein sequence encoding Compound 2 was back translatedusing a Saccharomyces cerevisiae codon usage table (Saccharomyces GenomeDatabase). To enable translation of the synthetic cDNA an additional ATGstart codon was added to the 5′ end and a TAA stop codon was added tothe 3′ end. The construct was inserted into HindIII and The EcoRI siteof the pYES2 shuttle vector comprising an ampicilline resistance gene,and the new construct was designated pYES0010, cf. FIG. 6. pYES0010 wassubsequently transformed into E. coli and subjected to ampicillinselection pressure. Positive clones were selected and sequenced.

[0292] Transformation into Yeast.

[0293] In order to make transform the pYES0010 into the yeast haploidINVSc1: MATa his3deltal leu2 trp1-289 ura3-52. Yeast were grown in YPDmedium (1% yeast extract, 2% peptone, 2% glucose, and 0.004% adeninesulfate) at 30 C. to saturation. 1 ml of culture was harvested fortransformations. 2 μl of 10 mg/ml carrier DNA was added and 1 μg ofpYES0010 was added and mixed. 0.5 ml (45% PEG 4000, 1M Li OAc, 0.5M EDTAand 1M Tris-HCl (pH 7.5) was added and mixed. Finally 20 μl 1M DTT wasadded and the mixture was incubated for 16 h at room temperature. Afterincubation the cells were heat shocked at 42 C. for 10 min and platedselective plates (6.7% yeast nitrogen base, 2% glucose, 20 μg/mladenine, 20 μg/ml arginine, 29 μg/ml isoleucine, 20 μg/ml histidine, 60μg/ml leucine, 20 μg/ml lysine,20 μg/ml tryptophan, 20 μg/ml methionine50 μg/ml phenylalanine 150 μg/ml valine, 30 μg/ml Tyrosine and 2.5%agar. Plates were incubated at 30 C. for 3 to 5 days until transformantsappear.

[0294] Expression and Purification of Compound 2.

[0295] Transformants were cultivated in selective media (6.7% Yeastnitrogen base, 2% glucose, 20 μg/ml adenine, 20 μg/ml arginine, 29 μg/mlisoleucine, 20 μg/ml histidine, 60 μg/ml leucine, 20 μg/ml lysine, 20μg/ml Tryptophan, 20 μg/ml methionine 50 μg/ml phenylalanine 150 μg/mlvaline, 30 μg/ml Tyrosine) for 1.5 days. The cells were harvested andresuspended in galactose induction medium (6.7% Yeast nitrogen base, 4%galactose, 20 μg/ml adenine, 20 μg/ml arginine, 29 μg/ml isoleucine, 20μg/ml histidine, 60 μg/ml leucine, 20 μg/ml lysine, 20 μg/ml Tryptophan,20 μg/ml methionine 50 μg/ml phenylalanine 150 μg/ml valine, 30 μg/mlTyrosine for 1 day. The cells were harvested and homogenized in 10 mMTris-HCl pH 7.5 containing protease inhibitors (Roche). The lysate wasclarified centrifugation at 20.000× g for 30 min. The supernatant wasloaded onto a Superdex 12 HR 10/30 column (Amersham Pharmacia Biotech)equilibrated with 10 mM Tris-HCl pH 7.5. The column was eluted in 50 Mmammonia bicarbonate buffer pH 8.0. Samples containing recombinantCompound 2 were pooled. The N-terminal methionine was removed bymethionine aminopeptidase and the samples were further purified on aHPLC Column.

[0296] HPLC Settings for Compound 2 Purification. HPLC column: KromasilRP C8; K 100-10-C8 nr. CER 2230. compound Temp: 22 C. Flow rate: 35ml/min HPLC solvents: A: 0.10% trifluoroacetic acid in water B: 0.10%trifluoroacetic acid in acetonitrile:water 90:10.

[0297] Compound 2 was eluted from the HPLC column with 0.10%trifluoroacetic acid in 20% to 80% Acetonitrile in 40 min.

[0298] 21. Injection Formulations of Peptide

[0299] Fixed dose formulations of peptide for intra venous injection areprepared by dissolving the peptide in sterile, isotonic saline, andstoring the resulting solution in glass ampoules filled with inert gasunder sterile conditions. Each dose of the peptide is stored dry inampoules or capped vials filled with inert gas. Multi-dose formulationsof peptide for intra venous injection are prepared by dissolving thepeptide in sterile, isotonic saline, storing the resulting solution incapped vials, if necessary adding preservative (for instance 0.1%parahydroxybenzoate, 1% benzyl alcohol or 0.1% chlorocresole). Exampleof multi-dose peptide formulation: Compound 2 12.25 mgSodiumdihydrogenphosphate 1.380 g Parahydroxybenzoate 0.1 g Aqua adinjectabile 100 ml

[0300] 22. Stability Experiments

[0301] In vitro stability studies with the present peptides and peptideconjugates in the presence of selected proteolytic enzymes are appliedas a tool for evaluating the protection of said peptides againstproteolysis in vivo. The aim of the experiments performed was to measureand compare the in vitro stability of Compounds 4, 5, 6 and 7 to that ofthe prior art compounds Compound(iii) H-(Gly⁸)-hGLP-1(7-36)-NH₂ (SEQ IDNO: 87) and hGLP-1(7-36)-NH₂ (SEQ ID NO: 114) in solutions of one ormore of the enzymes leucine aminopeptidase, carboxypeptidase A anddipeptidyl aminopeptidase IV at 37° C.

[0302] Materials and Apparatus for in vitro Stability

[0303] Water used was of highest quality obtained from a Milli-Q watertreatment system (Millipore, Bedford, Mass., USA). Acetonitrile (ACN)was of super gradient quality obtained from Labscan Ltd. (Dublin,Ireland). Trifluoracetic acid (TFA) 99.9%, dihydrogen phosphate(NaH₂PO₄), sodium hydroxide (NaOH) and all other chemicals used were ofanalytical grade. Leucine aminopeptidase (EC 3.4.11.1), CarboxypeptidaseA (EC 3.4.17.1) and Dipeptidyl peptidase (Dipeptidyl aminopeptidase IV,EC 3.4.14.5) were all obtained from Sigma (St. Louis, Mo., USA).Gradient HPLC analysis was done using a Hewlett Packard HP 1100 HPLCsystem consisting of a HP 1100 Binary Pump, a HP 1100 Autosampler, A HP1100 Column Thermostat and a HP 1100 Variable Wavelength Detector.Hewlett Packard Chemstation for LC software (Rev. A.06.01) was used forinstrument control and data acquisition. A Vydac 238TP54 (150×4.6 mmI.D.) column packed with 5 m, C18, 300 particles was used with theinstrument. A SHT200D block heater from Stuart Scientific was used forheating of the peptide/enzyme solutions during the stabilityexperiments. The degradation of the test compounds was studied at 37° C.in 50 mM phosphate buffer solutions of pH 7.4 containing leucineaminopeptidase (25 U/ml) or carboxypeptidase A (1 U/ml) or 100 mMammoniumbicarbonate buffer of pH 8.0 containing dipeptidylaminopeptidase IV (0.5 U/ml). Experiments were initiated by addition ofan aliquot (100 l) of a stock solution (1 mg/ml) of the peptide in waterto 900 l preheated enzyme solution in an Eppendorf microvial giving aninitial concentration of 0.1 mg/ml (˜1.7·10−5-1.8·10−5 M) of thepeptide. The peptide/enzyme solution was kept at 37° C. and atappropriate time intervals samples of 100 l were withdrawn from thepeptide/enzyme solution and mixed thoroughly with 20 125% TFA inacetonitrile in order to stop the enzymatic degradation process. Theinactivated samples were transferred to autosampler vials and analysedfor content of intact test compound by HPLC as described below.Half-lives (t½) for the test compounds in enzyme solutions werecalculated from plots of natural logarithm to the residual concentration(i.e. HPLC peak heights) against time using the formula:t_(1/2)=1/k_(obs)In(2), where k_(obs) is the apparent first-order rateconstant for the observed degradation.

[0304] HPLC Analysis

[0305] Samples from the stability experiments performed as describedabove were analysed by gradient HPLC analysis using the instrumentationdescribed above and the following experimental conditions. Columntemperature: 30° C. Injection volume: 10 l Mobile phase A: 0.1% TFA inwater Mobile phase B: 0.085% TFA in acetonitrile (ACN) Gradient: 32-52%B in 21 min Detection: UV at 215 nm

[0306] The experimental results obtained from the individual stabilityexperiments are shown in Table I below. It appears from the table thatthe half life of the compounds of the invention is considerably extendedin solution with all enzymes tested. TABLE 1 Test Compound EnzymeSolution Half-life Compound No. Name Enzyme Conc. (t½) Compound 5H-(Gly⁸)hGLP 1(7-36)- LAP  25 U/ml   >3 days Lys(Palm) -Lys₆-NH₂ (SEQ IDNO: 153) CPA   1 U/ml   >2 days DPP IV 0.5 U/ml  440 min Compound 7H-(Gly⁸, Lys²⁶(Palm))- LAP  25 U/ml 1150 min hGLP-1(7-36)-Lys₆-NH₂ (SEQID NO: 103) CPA   1 U/ml 1058 min DPP IV 0.5 U/ml  526 min Compound 6H-(Gly⁸, Lys³⁴(Palm))- LAP  25 U/ml ˜1.5 day hGLP-1(7-36)-Lys₆-NH₂ (SEQID NO: 90) CPA   1 U/ml   >1 day DPP IV 0.5 U/ml  177 min GLP-1H-hGLP-1(7-36)-NH₂ LAP  25 U/ml  152 min (SEQ ID NO: 114) CPA   1 U/ml 48 min DPP IV 0.5 U/ml   2.0 min Compound 4 H-(Gly⁸)-hGLP-1(7-36)- LAP 25 U/ml ˜1.5 day Lys₆-NH₂ (SEQ ID NO: 88) CPA   1 U/ml  145 min DPP IV0.5 U/ml  292 min Compound(iii) H-(Gly⁸)hGLP-1(7-36)- LAP  25 U/ml  693min NH₂ (SEQ ID NO: 87) CPA   1 U/ml  127 min DPP IV 0.5 U/ml  56 min

[0307] 23. In Vitro Stability Studies of Compound (iii) and Compound 4in Rat Plasma

[0308] The degradation of the two test compounds and in heparinstabilised rat (Sprague-Dawley) plasma was followed by the combinationof solid phase extraction and LC-MS. The degradation was followed for720 minutes in plasma. The half-life of Compound (iii) was found to be238 min. in rat plasma. This finding was compared with the half-life ofCompound 4, which was found to be 466 min. in rat plasma.

[0309] Materials And Methods

[0310] Blank rat plasma in sodium heparin (5000 units/mL) were obtainedfrom Harlan Sera Lab Ltd. (Loughborough, UK). Test Substances andSolutions The test substances used in the study are listed in the tablebelow. For the in vitro experiments a stock solution of 100 μg/mlmilli-Q water was used (corresponding to 26.0 μM Compound (iii)H-(Gly⁸)-GLP-1(7-36) (SEQ ID NO: 87)-NH₂ or 17.8 μM Compund 4).Substance Name Batch No. Average Mw. Peptide Content Compound (iii) ZP7, 73-1F 3284 g/mol 85% Compound 4 ZP 7, 69-1C 4053 g/mol 72%

[0311] The LC-MS analysis was performed on an HP 1100 instrumentconsisting of an on-line degasser, a quaternary gradient pump, an autosampler, a column oven, Hewlett Packard (Wilmington, Del., USA) incombination with a Quattro Ultima mass spectrometer from Micromass(Altrincham, UK). Both the LC and MS were controlled by MassLynx 3.3software. The LC separations prior to MS detection were performed on aVydac 218MS52 (2.1×250 mm) column (Hesperia, Calif., USA).

[0312] The initial plasma volume was 1000 μl (37° C.). From the initialplasma volume, 100 μl was transferred to a 0.75 ml HPLC vial (used asblank), mixed with 560 μl extraction solution (MeCN:0.18 M ammoniumcarbonate pH 9.5 (6:94 v/v), 4 C.) and extracted by Solid PhaseExtraction using ASPEC XL4 Robot. A volume of 100 μl stock solution wasadded to the remaining 900 μl plasma, mixed thoroughly and incubated at37 C. (corresponding to an initial concentration of 10 μg of the testcompounds/ml). At each time point (0.2, 60, 120, 180, 240, 360, 480, 662and 720 min., respectively) 100 μl of the drug containing plasma wascollected, mixed with 560 μl ice cold extraction solution andimmediately extracted by SPE as described above. The extracted plasmasamples were analysed by LC-MS. The LC-MS analysis were performed on anHP 1100 series LC in combination with a Quattro Ultima II triplequadrupole MS instrument.

[0313] The samples were kept at 18° C. in the autosampler tray prior toinjection of 10 μl. The separations were performed at 30° C. on a Vydac218MS52 (2.1×250 mm) LC column using a linear gradient from 15 to 50% Bwithin 14 min. at a flow rate of 250 μl/min. 0.1% formic acid in waterwas used as mobile phase A and 0.1% formic acid in MeCN as mobile phaseB. Compound 4 and Compound (iii) were detected by single ion recording(SIR) using the 6H+(m/z=676.7) and 4H+(m/z=822.1) ion species,respectively. The cone voltage for the analysis of compound (iii) andCompound 4 was set to 100 and 70 V, respectively.

[0314] The in vitro stability of Compound (iii) and Compound 4 have beeninvestigated in rat plasma by LC-MS. The degradation of the twocompounds were followed for 720 min. and the results were plotted as thenatural logarithm of the peak area vs. time. The degradation rates(k_(obs)) of the compounds were found as the slope after linearregression, and the half-life (T½) was found as In 2/k_(obs). Theresults from the experiment are listed below.

[0315] Degradation Study over 720 Minutes in Rat Plasma Compound T½(min) k_(obs) (min⁻¹) r² Compound (iii) 238.4 0.0029 0.9785 Compound 4466.1 0.0015 0.8596

[0316] The conclusion of the experiment is therefore that the provisionof a C-terminal Lys₆ (SEQ ID NO: 9) peptide conjugation to the(Gly⁸)hGLP-1(7-36) (SEQ ID NO: 87) sequence results in a two foldincreased stability in rat plasma.

[0317] 24. Single Dose Effect of oral and parenteral administration ofCompound 5 on Blood Glucose Levels in Diabetic ob/ob Mice.

[0318] The compounds of the invention possess blood glucose loweringproperties. This was examined using Compound 5 to test the effect onblood glucose (BG) levels in the ob/ob mutant mice after intraperitoneal(i.p.) and peroral (p.o.) administration. Compound 5 reduced BG levelsin diabetic mice in a dose of 110 μg/mouse when administered i.p.Likewise p.o. administration of Compound 5 elicited a similar decreasein BG levels in a dose of 1100 μg/mouse, but not at lower doses.

[0319] Experimental

[0320] Forty female diabetic ob/ob mice (Umea strain, Bomholtgaard),which are obese due to a dominant mutant leptin (Tomita, T., Doull, V.,Pollock, H. G., and Krizsan, D. 1992. Pancreatic islets of obesehyperglycemic mice (ob/ob). Pancreas 7: 367-75) were housed (3mice/cage) under controlled ambient conditions following a 12:12-hlight:dark cycle and fed standard Altromin no 1324 diet with free accessto tap water. At arrival the animals were 8 weeks of age. The mice wereallowed 2 weeks of acclimatization before experiments were initiated. Atthe time of experiment the mice were 13 weeks old with a body weight of41.8±3.2 g (mean±SD; n=42). Handling of the mice one and three daysbefore the experiment was performed in order to reduce stress-induced BGexcursions. On the day of the experiment, blood was taken from the tipof the tail 2-3 hours after the light was turned on. A single drop ofblood (<5 μl) was dropped on the glucose strip for analysis and measuredby an Elite Autoanalyser, Bayer, Denmark. Whole blood glucose (BG)concentration was analysed by the immobilised glucose oxidase method.Blood glucose levels varied between normoglycaemia and severehyperglycaemia (range: 3.6-15.6 mM; mean±SD: 9.4±3.3 mM; n=42). Sixanimals with BG<5.8 mM were excluded from the study (total n=36). Theremaining animals were stratified based on their BG levels in order toensure that the mean BG was similar among groups. One hour after theinitial control blood sampling, drugs were administered and BG wasmeasured at t=60 min, t=120 min, t=240 min, t=480 min.

[0321] Peptides and Other Materials

[0322] Compound 5 (batch nr. ZP 3.12 fraction 1-2. Purification) wassynthesised by the Department of Chemistry, Zealand Pharmaceuticals. Thepeptide was dissolved in sterile isotonic NaCl shortly before dosing andgiven in a volume of 0.2 ml. The same solutions were used for both p.o.and i.p. administration. For each animal, a data log sheet was filledout at the time of each blood sampling.

[0323] Drug Administration

[0324] Animals were administered with Compound 5, and the maximum dosewas 1100 μg/mouse and the lowest dose was 1.1 μg/mouse. As a negativecontrol, saline was administered p.o. and as positive control the testcompound was given i.p. in a dose of 110 μg/mouse.

[0325] During control conditions, BG levels in non-fasted ob/ob micewere similar in all groups (individual group data not shown), but withingroups, there was a great scatter on BG levels (BG range for allanimals: 5.8-15.6 mM). Therefore, to correct for the varying degree ofhyperglycemia, results are expressed as the relative difference frombaseline (% control). Intraperitoneal administration of 110 μg Compound5 produced a sustained decrease in BG that reached nadir at 1-2 hrsafter administration of the compound. No changes were observed in salinetreated animals. In most groups (5/6), BG increased between 4 and 8 hrsafter drug administration. Compound 5 reduced the BG levels in a dose of110 μg/mouse when administered i.p. in diabetic ob/ob mice (data notshown). The antidiabetic effect was observed after 60 minutes and wasmaximal 2-4 h after administration of the compound. Furthermore, along-lasting effect (>8 hours) suggests that Compound 5 has a longerduration of action than the notoriously short-acting native GLP-1(Bailey, C. J. & Flatt, P. R. 1987. Glucagon-like peptide-1 and theentero-insular axis in obese hyperglycaemic (ob/ob) mice. Life Sci, 40,521-5). The dose 1100 μg/mouse p.o. elicited a similar decrease in BG asobserved in animals treated with 110 μg i.p.

[0326] We have shown that Compound 5 effectively lowers BG levels indiabetic ob/ob mice following i.p. administration of 110 μg/mouse thecompound. A similar effect is seen after 1100 μg/mouse of Compound 5when given by the oral route. This suggests that the compound isabsorbed from the gastrointestinal tract.

[0327] 25. In Vivo Studies with

[0328] Compound 1 (des Pro³⁶-exendin-4(1-39)-NH₂ (SEQ ID NO:101)),

[0329] Compound 2 (des Pro³⁶-exendin-4(1-39)-Lys₆-NH₂ (SEQ ID NO:93)),

[0330] Compound (iii) (Gly⁸-GLP1-(7-36)(Human)-NH₂ (SEQ ID NO:87)),

[0331] Compound 4 (Gly⁸-GLP1-(7-36)(Human)-Lys₆-NH₂ (SEQ ID NO:88)) and

[0332] Compound 5 (Gly⁸Lys³⁷(palmitoyl)-GLP1-(7-36)(Human)-Lys₇-NH₂ (SEQID NO:89))

[0333] Various concentrations of each peptide are administered orallyand intraperitoneally to ob/ob mice to determine if these compoundsaffect blood glucose levels. The experimental conditions used were thesame as described in Example 24.

[0334] Peptides and other Materials

[0335] Des Pro³⁶-exendin-4(1-39)-NH₂ (Compound 1, SEQ ID NO: 101) andthe same peptide, but with an additional sequence, Lys₆, attached at theC-terminal, des Pro³⁶-exendin-4(1-39)-Lys₆-NH₂ (Compound 2, SEQ IDNO:93), Gly⁸-GLP1-(7-36)(Human)-NH₂ (Compound (ii), SEQ ID NO:87)and thesame peptide, but with an additional sequence, Lys₆, attached at theC-terminal, Gly⁸-GLP1-(7-36)(Human)-Lys₆-NH₂ (Compound 4, SEQ ID NO:88)and Gly⁸Lys³⁷(palmitoyl)-GLP1-(7-36)(Human)-Lys₇-NH₂ (Compound 5, SEQ IDNO:89) are synthesized using methods described above. Solutions areprepared on the morning of dosing, immediately before the animals areadministered. The same solutions are used for both peroral andinterperitoneal administration. All peptides are dissolved in sterileisotonic NaCl and given in a volume of 0.2 ml. All experiments arecarried out in the same mice to compare the active doses of the peptidesshown in Table 2. Blood sampling is performed as described above and theanimals are administered with the doses shown in Table 3. As negativecontrol, saline is administered perorally. Results are shown in Table 4.TABLE 2 Number Compound Compound 1 des Pro³⁶-exendin-4(1-39)-NH₂ (SEQ IDNO: 101) Compound 2 des Pro³⁶-exendin-4(1-39)-Lys₆-NH2 (SEQ ID NO: 93)Compound (ii) Gly⁸-GLP1-(7-36)(Human)-NH2 (SEQ ID NO: 87) Compound 4Gly⁸-GLP1-(7-36)(Human)-Lys₆-NH2 (SEQ ID NO: 88) Compound 5Gly⁸Lys³⁷(palmitoy1)-GLP1-(7-36)(Human)-Lys₇-NH₂ (SEQ ID NO: 89)

[0336] TABLE 3 Group 5 Dose Group 1 Group 2 Group 3 Group 4 peroral DoseDose Dose Dose μl/mouse Group 6 peroral peroral peroral peroral IsotonicDose i.p Compound μg/mouse μg/mouse μg/mouse μg/mouse saline μg/mouseCompound 1 400 40 4 0.4 200 μl 40 Compound 2 1000 100 10 1 200 μl 100Compound (ii) 1000 100 10 1 200 μl 100 Compound 4 1000 100 10 1 200 μl100 Compound 5 1000 100 10 1 200 μl 100

[0337] Group data were summarised as the mean±SEM of the individualresults in each treatment group. In order to analyse the effects of thecompounds, the absolute and the relative (% of t=0) difference frombaseline was calculated for each time point. TABLE 4 0 1 hour 2 hours 4hours Compound 1-saline 100 103 107 92 Compound 1-400 μg po 100 93 88 93Compound 1-40 μg po 100 89 89 91 Compound 1-4 μg po 100 105 88 91Compound 1-0.4 μg po 100 106 103 100 Compound 1-40 μg ip 100 68 69 74Compound 2-saline 100 100 112 114 Compound 2-1000 μg po 100 67 69 64Compound 2 100 μg po 100 78 71 72 Compound 2 10 μg po 100 86 72 72Compound 2 1 μg po 100 112 101 96 Compound 2 100 μg ip 100 75 67 63Compound (ii)-saline 100 95 87 100 Compound (ii)-1000 μg po 100 87 10594 Compound (ii)-100 μg po 100 118 111 92 Compound (ii)-10 μg po 100 10194 104 Compound (ii)-1 μg po 100 94 89 96 Compound (ii)-100 μg ip 100 7060 81 Compound 4-saline 100 102 94 79 Compound 4-1000 μg po 100 128 7278 Compound 4-100 μg po 100 72 70 58 Compound 4-10 μg po 100 98 95 81Compound 4-1 μg po 100 99 89 84 Compound 4-100 μg ip 100 83 58 56Compound 5-saline 100 90 86 103 Compound 5-1000 μg po 100 73 75 67Compound 5-100 μg po 100 97 140 107 Compound 5-10 μg po 100 90 120 126Compound 5-1 μg po 100 111 133 114 Compound 5-100 μg ip 100 63 50 52

[0338] These results show that all tested compounds have an effect inlowering blood glucose levels. The effect is most pronounced whenCompound 1 is given intraperitoneally whereas the effect of 1000 μg poof Compound 2 is comparable to the effect of 100 μg ip of Compound 2.The potency of Compound 1 (des Pro³⁶-exendin-4(1-39)-NH₂, SEQ ID NO:101)and Compound 2 (des Pro³⁶-exendin-4(1-39)-Lys₆-NH₂, SEQ ID NO:93) whengiven intraperitoneally is shown to be very similar toexendin-4(1-39)-NH₂ (SEQ ID NO: 102) (Compound (i)) itself (data notgiven) administered in the same way.

[0339] For Compound 1, des Pro³⁶-exendin-4(1-39)-NH₂ (SEQ ID NO:110),there is no effect in lowering blood glucose levels up to a dose of 400μg/mouse when the compound is administered perorally, whereas for thesame compound with the addition of the Lys₆ fragment there is activityseen at a dose of 10 μg/mouse. This indicates that the minimum effectiveoral dose of the des Pro³⁶exendin-4(1-39)-Lys₆-NH₂ (SEQ ID NO:93) is atleast 40 times lower than for des Pro³⁶-exendin-4(1-39)-NH₂ (SEQ IDNO:101).

[0340] These results show that the attachment of the sequence Z has nosignificant effect on the potency of the various peptides whenadministered interperitoneally while significantly enhancing the potencyof the compound when administered perorally.

[0341] 26. Bioavailability of Compound 4 and Compound (iii) afterGastro-Intestinal Delivery in Duodenum in Conscious Rats.

[0342] Various peptide based GLP-1 analogues have been developed forparenteral use, but none of these substances has been pharmacologicallyeffective after oral administration [Hoist, J. J.: Enteroglucagon. AnnuRev Physiol, 59:257-271, 1997]. It was decided to examine the absorptionof the test compound from the duodenum in conscious rats. Compound (iii)(Gly⁸)hGLP-1(7-36)-NH₂ (SEQ ID NO: 87) was used as reference.

[0343] Chemicals and Reagents

[0344] Blank rat plasma in sodium heparin (5000 units/mL) were obtainedfrom Harlan Sera Lab Ltd. (Loughborough, UK). OASIS™ HLB solid phaseextraction columns, 1 cc, 30 mg sorbent, were obtained from Waters(Milford, Mass., USA) and ISOLUTE C18 (EC), 1 cc, SPE columns wereobtained from IST (Mid Glamorgan, U.K.). The LC/MS analysis wasperformed on a HP 1100 instrument consisting of an on-line degasser, abinary gradient pump, an auto sampler, a column oven, Hewlett Packard(Wilmington, Del., USA) in combination with a Quattro Ultima massspectrometer from Micromass (Altrincham, UK) both the LC and MS werecontrolled by MassLynx 3.3 software. The LC separations prior to MSdetection were performed on a Vydac 218MS52 (2.1×250 mm) column(Hesperia, Calif., USA).

[0345] Drugs and Dose Levels:

[0346] Compound 4 (batch No. ZP 7.97-5-F, 4053 g/mol) and Compound (iii)(batch No. ZP 7.73-2-G, 3854 g/mol) were synthesised in-house using theFmoc strategy. The identification was performed by mass spectrometry andthe purity of both batches was determined by RP-HPLC to 97 and 99.7% forthe test compounds, respectively. The peptide content of the batcheswere 72% and 80% for ZP 7.97-5-F and ZP 7.73-2-G, respectively. Thepeptides were dissolved in pyrogen free isotonic saline and doses of1.000 or 10.000 nmol/kg administered through the intra duodenal catheterin a volume of 100 μl.

[0347] Animals:

[0348] Fourteen Sprague-Dawley rats weighing 250 to 350 g. were used forthe experiment. The rats were anaesthetised with Hypnorm®-Dormicum® s.c.and a catheter was inserted into the femoral artery for arterial bloodsampling. An additional catheter was inserted into the duodenum via anincision in the ventricle. Before the experiment was started, the ratswere allowed to recover for one week after the operation. The operatedrats were conscious at the day of the experiment. In order to establishwhether the intra duodenal catheters were situated in the duodenum, anautopsy was performed on the rats immediately after the experiment.

[0349] Sample Treatment:

[0350] Blood samples were collected at t=−5, 5, 10, 15, 20, 40, and 60min. The blood was collected in EDTA containing ice-chilled tubes andimmediately centrifuged at 4° C. for 5 min (4.000×g). Plasma (250 μl)was transferred to ice-chilled 0.75 ml PLC vials containing 250 μlextraction solution (MeCN: 0.18 M Ammonium Carbonate pH 9.5, 10:90 v/v).The plasma samples were stored at −20?C until SPE and LC/MS analysis.

[0351] Solid Phase Extraction:

[0352] The drug containing plasma samples (400 μl) were loaded ontosolid phase extraction columns preconditioned with 950 Pl MeCN followedby 950 μl water. The columns were washed with 950 Pl 2% TFA in waterfollowed by an equal volume of 2% TFA in MeCN:water (20:78 v/v). Theanalytes were eluted with 500 μl 2% TFA in MeCN:water (60:38 v/v) andanalysed by LC/MS.

[0353] LC/MS

[0354] The samples were kept at 18° C. in the auto sampler tray prior toinjection of 20 to 50 μl onto the LC column (Vydac 218MS52 (2.1×250 mm).The separations were performed at 30° C. using a flow rate of 250 μl/minand a gradient according to Table 1. Both the test compound and thereference drug were detected by single ion recording (SIR) using them/z=676.7 and the m/z=1095.2 and 821.8 ion species, respectively. Allinstrument conditions were controlled by MassLynx software ver. 3.3software. Compound Gradient Compound 4 Initial: 15% B, 0-14 min; 15-50%B, 14-15 min; 50-15% B and 15-20 min 15% B. Compound (iii) Initial: 25%B, 1-1.5 min; 25-30% B, 1.5-10 min; 30-40% B, 10-10.5 min; 40-90% B,11.5-12 min; 90-25% B, and 12-17 min 25% B.

[0355] The gradient used for the analysis of the test compounds using0.1% formic acid in water or MeCN as Mobile phase A or B, respectively.

[0356] The plasma samples were analysed as described under materials andmethods. The bioavailability of Compound 4 was examined in doses of1.000 (n=4) and 10.000 (n=5) nmol/k, whereas Compound (iii) was onlystudied in a dose of 10.000 (n=5) nmol/kg. At all the investigated timepoints the concentration of Compound (iii) was below the detection limit(approx. 0.5 nM), the exact bioavailability could therefore not beestimated. In contrast, Compound 4 was detected in the plasma samplesfrom two out of four rats after intra duodenal administration of 1.000nmol/kg and in four out of five rats following administration of 10.000nmol/kg.

[0357] 27. In Vivo Pharmacokinetics of Compound 1, Compound 2, Compound4, and Compound (iii) after i.v. Administration to Rabbits and Pigs

[0358] We have shown an increased in vitro stability of the GLP-1agonist Compound 4 when compared to the reference drug Compound (iii) inrat plasma. In order to establish whether this effect is sustained invivo, the pharmacokinetic parameters of the two compounds are examinedin rabbits. Using the same experimental conditions these parameters werealso measured for Compounds 1 and 2 in rabbits and using similarconditions in pigs.

[0359] Chemicals and Reagents

[0360] Blank rabbit plasma in sodium heparin (5000 units/mL) wereobtained from Harlan Sera Lab Ltd. (Loughborough, UK). OASIS™ HLB solidphase extraction columns, 1 cc, 30 mg sorbent, were obtained from Waters(Milford, Mass., USA) and ISOLUTE C18 (EC), 1 cc, SPE columns wereobtained from IST (Mid Glamorgan, U.K.). The LC/MS analysis wasperformed on a HP 1100 instrument consisting of an on-line degasser, abinary gradient pump, an auto sampler, a column oven, Hewlett Packard(Wilmington, Del., USA) in combination with a Quattro Ultima massspectrometer from Micromass (Altrincham, UK) both the LC and MS werecontrolled by MassLynx 3.3 software. The LC separations prior to MSdetection were performed on a Vydac 218MS52 (2.1×250 mm) column(Hesperia, Calif., USA).

[0361] Drugs and dose levels:

[0362] Compound 4 (batch No. ZP 7.97-5-F, 4053 g/mol) and Compound (iii)(batch No. ZP 7.73-2-G, 3854 g/mol) were synthesised in-house using theFmoc strategy. The identification was performed by mass spectrometry andthe purity of both batches were determined by RP-HPLC to 97 and 99.7%for the test compounds, respectively. The peptide content of the batcheswere 72% and 80% for ZP 7.97-5-F and ZP 7.73-2-G, respectively. Thepeptides were dissolved in pyrogen free isotonic saline and bothpeptides were administered i.v. to rabbits and rats using a dose of 1000nmol/kg.

[0363] Rabbits:

[0364] Fifteen New Zealand White rabbits weighing 2.5 to 3.0 kg wereused for the experiment. On the day of the experiment, the rabbits wereanaesthetised with Hypnorm® i.m. followed by Dormicum® i.v. Catheterswere inserted into the femoral vein and artery for i.v. administrationof drugs and arterial blood sampling. The rabbits stayed unconsciousthroughout the experiment.

[0365] Sample Treatment:

[0366] Blood samples were collected at t=1, 3, 5, 10, 15, 20, 30, 40,60, 90, 120, 150, 180, and 240 min. The blood was collected in EDTAcontaining ice-chilled tubes and immediately centrifuged at 4 C. for 5min (20.000×g). Plasma (250 μl) was transferred to ice-chilled 0.75 mlPLC vials containing 250 μl extraction solution (MeCN: 0.18 M AmmoniumCarbonate pH 9.5, 10:90 v/v). The plasma samples were stored at −20 C.until SPE and LC/MS analysis.

[0367] Solid Phase Extraction:

[0368] The drug containing plasma samples (400 μl) are loaded ontoOASIS™ HLB (Compound 4) or ISOLUTE™ (Compound (iii)) solid phaseextraction columns preconditioned with 950 μl MeCN followed by 950 μlwater. The columns are washed with 950 μl 2% TFA in water followed by anequal volume of 2% TFA in MeCN:water (20:78 v/v). The analytes areeluted with 500 μl 2% TFA in MeCN:water (60:38 v/v) and analysed byLC/MS.

[0369] LC/MS

[0370] The samples were kept at 18 C. in the auto sampler tray prior toinjection of 20 to 50 μl onto the LC column (Vydac 218MS52 (2.1×250 mm).The separations were performed at 30° C. using a flow rate of 250 μl/minand a gradient according to the table below. Both the test compound andthe reference drug are detected by single ion recording (SIR) using them/z=676.7 and the m/z=1095.2 and 821.8 ion species, respectively. Allinstrument conditions were controlled by MassLynx software ver. 3.3software. Compound Gradient Compound 4 Initial: 15% B, 0-14 min; 15-50%B, 14-15 min; 50-15% B and 15-20 min 15% B Compound (iii) Initial: 25%B, 1-1.5 min; 25-30% B, 1.5-10 min; 30-40% B, 10-10.5 min; 40-90% B,11.5-12 min; 90-25% B, and 12-17 min 25% B.

[0371] The gradient used for the analysis of the test compounds using0.1% formic acid in water or MeCN as Mobile phase A or B, respectively.

[0372] The plasma samples were analysed as described under materials andmethods and the plasma concentration (C_(pl)) plotted versus time in asemi log diagram. The plasma concentration were followed for three hoursin rabbits, whereas the limited blood volume of rats restricted theblood sampling in this specie to one hour. The C_(pl) vs. time curvesfrom the individual rabbits were fitted to a two-compartment open model(figure not shown) using 1/y² weighted least squares in WinNonlin 3.1(Pharsight Corp. (Mountain View, Calif.)). The pharmacokinetic constantsobtained from the data analysis are listed in Table 5 and thedegradation kinetics in rabbit after i.v. injection of 1 μmol/kg ofCompound 4 and Compound (iii), respectively, is shown in FIG. 4. TABLE 5In vivo kinetics in rabbits and pigs ** Comp. (iii) Comp. 4 Comp. 1Comp. 2 Comp. 2 ** (n = 7) (n = 8) (n = 5) (n = 5) (n = 2) ParameterMean Mean Mean Mean Mean T_(½), α min 2.3 6.8 4.4 11 16 T_(½), β min10.8 28.0 23 69 252

[0373] Table 5: The pharmacokinetic constants were obtained from rabbitswhen the C_(pl) vs. time curves was fitted mathematically. The compoundswere administered iv in a concentration of 1000 nmol/kg. T_(1/2) valuesare given in minutes (min) for the α and β phase. Statistics: two-tailedt-test assuming samples with unequal variances showed p<0.001 for allmeasured parameters. In conclusion the T½ value for Compound 4 isapproximately three times the value for the reference Compound (iii)and, likewise, the T½ value for Compound 2 is approximately three timesthe value calculated for Compound 1 which represents the unconjugatedequivalent.

[0374] 28.Glucose Tolerance Test of Compounds 2, 14-16, 18 and 19compared to Compound (i)

[0375] Male diabetic db/db mice (M&B, Bomholdtgaard, Ll. Skensved,Denmark) are used. This well-described mouse model has inheritedmalfunctions of the glucose metabolism due to a mutation in the leptinreceptor. Like human patients with uncontrolled non-insulin demandingdiabetes mellitus (NIDDM), homozygous db/db mice experience polydipsia,polyuria and glycosuria and gain weight during their first 3 months oflife despite their hyperglycaemic stage. However, in this model thehyperglycaemia is associated with progressive pancreatic islet atrophywith possible ketosis and death at 6-8 months of age. Thus, attentionshould be paid to the progression and status of their disease state.Therefore, preferably only db/db mice less than 16 weeks old should beused for drug testing og GLP-1 analogues.

[0376] All animals are acclimatised for at least one week and handleddaily for two days prior to the first oral glucose tolerance test(OGTT). Furthermore, to reduce stress-induced glucose excursions, theanimals should be subjected to at least one OGTT without compound asdescribed below prior to the experiment. Due to the great scatter ofglucose tolerance among diabetic mice, the animals are stratified by anOGTT prior to their first use.

[0377] Peptides

[0378] Peptides are dissolved in 0.1 M phosphate-buffered saline (PBS)with 0.1% bovine albumin where pH is adjusted to 7.4 by adding 5 M NaOH.All solutions are prepared fresh on the morning immediately before theexperiment. Vehicle treated animals are given PBS with 0.1% albuminalone.

[0379] Glucose Tolerance Test and Dosing

[0380] Before the oral glucose tolerance test, the animals are fastedfor 17 hours (from 4 p.m. until 9 a.m. the following morning). Beginningat 9.00 a.m. blood is taken from the tail tip (t=−15 min) and bloodglucose is measured. The whole blood glucose (mM) concentration isanalysed by the immobilised glucose oxidase method using a drop of blood(<5 μL, Elite Autoanalyser, Bayer, Denmark) following the manufacturer'smanual. Animals with severe diabetes (>10 mM) are excluded. Immediatelyafter the initial blood sample, the animals receive an intraperitoneal(i.p.) injection of vehicle or a dose of antidiabetic compound.Injection volume is 200 μl/50 g body weight in all groups. Fifteenminutes after i.p. administration of the substance an oral dose of 1g/kg glucose (Sigma, St. Louis) dissolved in water (200 μl/50 g bodyweight) is given, and the animals are returned to their home cages(t=0). Blood glucose levels are measured at t=30 min, t=60 min, t=120min and t=240 min. The animals are fasted during the observation period.For each animal a data log sheet was filled in at the time of each bloodsampling.

[0381] Calculations and Statistics

[0382] In order to analyse the effects of the compounds, the absoluteand the relative difference from baseline (t=0) are calculated for eachtime point. The area under the curve for the whole experiment (AUC₀₋₂₄₀min) is determined using the trapezoid method. On the day ofstratification, the mice are distributed in order to ensure that theglucose tolerances are similar in all groups. However, to correct forthe progression of the diabetes with time, a vehicle treated controlgroup is tested on each day of experiment and the response to drugs areexpressed relative to response observed in vehicle-treated time-controlanimals. Dose-response curves for each substance are plotted, cf. FIG.5, and the effect of drug relative to responses obtained duringtreatment with vehicle are analysed using an ANCOVA analysis (analysisof covariance). Treatment (drug or vehicle) is considered theindependent variable, AUC₀₋₂₄₀ min expressed as per cent response invehicle-treated time-control mice is the dependent variable, and drugdose is defined as covariate. Post-hoc analysis is performed usingFisher's Least Significant test. Differences are considered significantat the 0.05 level. Statistical analyses were performed using Statisticaversion 5.5 for Windows NT, StatSoft, Tulsa, Okla., U.S.A. The doseresponse curves shown in FIG. 5 clearly shows that all tested compoundsexhibit a glucose lowering effect comparable to that of the referencedrug.

[0383] 29. Effects of Compound 2 and Compound (i) on OGGT in db/db miceFIG. 7 is a plot of AUC for Compound 2 and Compound (i) in an OGTTperformed using the same experimental conditions as described in Example28. The figure shows that the blood glucose lowering effect of Compound2 is the same as the effect of the prior art compound (iii).

[0384] 30. Long term effects of Compound 2, 100 nmol/kg i.p. on the oralglucose tolerance test. (OGTT) when administered up to 24 hours beforethe OGTT

[0385] This experiment uses the maximal dose of 100 nmol/kg i.p. indb/db mice and otherwise, the same experimental conditions as describedin Example 28 are used. Results are shown in FIG. 8 and the conclusionof the experiment is that the duration of action of Compound 2 is up to18 hours in db/db mice.

[0386] The invention described and claimed herein is not to be limitedin scope by the specific embodiments herein disclosed, since theseembodiments are intended as illustrations of several aspects of theinvention. Any equivalent embodiments are intended to be within thescope of this invention. Indeed, various modifications of the inventionin addition to those shown and described herein will become apparent tothose skilled in the art from the fore-going description. Suchmodifications are also intended to fall within the scope of the appendedclaims. Various references are cited herein, the disclosure of which areincorporated by reference in their entireties.

1 153 1 4 PRT Artificial Sequence Description of Artificial Sequencesynthetic peptide sequence 1 Lys Lys Lys Lys 1 2 5 PRT ArtificialSequence Description of Artificial Sequence synthetic peptide sequence 2Lys Lys Lys Lys Lys 1 5 3 5 PRT Artificial Sequence Description ofArtificial Sequence synthetic peptide sequence 3 Xaa Lys Lys Lys Lys 1 54 5 PRT Artificial Sequence Description of Artificial Sequence syntheticpeptide sequence 4 Lys Xaa Lys Lys Lys 1 5 5 5 PRT Artificial SequenceDescription of Artificial Sequence synthetic peptide sequence 5 Lys LysXaa Lys Lys 1 5 6 5 PRT Artificial Sequence Description of ArtificialSequence synthetic peptide sequence 6 Lys Lys Lys Xaa Lys 1 5 7 5 PRTArtificial Sequence Description of Artificial Sequence synthetic peptidesequence 7 Lys Lys Lys Lys Xaa 1 5 8 6 PRT Artificial SequenceDescription of Artificial Sequence synthetic peptide sequence 8 Lys LysLys Lys Lys Lys 1 5 9 6 PRT Artificial Sequence Description ofArtificial Sequence synthetic peptide sequence 9 Xaa Lys Lys Lys Lys Lys1 5 10 6 PRT Artificial Sequence Description of Artificial Sequencesynthetic peptide sequence 10 Lys Xaa Lys Lys Lys Lys 1 5 11 6 PRTArtificial Sequence Description of Artificial Sequence synthetic peptidesequence 11 Lys Lys Xaa Lys Lys Lys 1 5 12 6 PRT Artificial SequenceDescription of Artificial Sequence synthetic peptide sequence 12 Lys LysLys Xaa Lys Lys 1 5 13 6 PRT Artificial Sequence Description ofArtificial Sequence synthetic peptide sequence 13 Lys Lys Lys Lys XaaLys 1 5 14 6 PRT Artificial Sequence Description of Artificial Sequencesynthetic peptide sequence 14 Lys Lys Lys Lys Lys Xaa 1 5 15 6 PRTArtificial Sequence Description of Artificial Sequence synthetic peptidesequence 15 Xaa Xaa Lys Lys Lys Lys 1 5 16 6 PRT Artificial SequenceDescription of Artificial Sequence synthetic peptide sequence 16 Xaa LysXaa Lys Lys Lys 1 5 17 6 PRT Artificial Sequence Description ofArtificial Sequence synthetic peptide sequence 17 Xaa Lys Lys Xaa LysLys 1 5 18 6 PRT Artificial Sequence Description of Artificial Sequencesynthetic peptide sequence 18 Xaa Lys Lys Lys Xaa Lys 1 5 19 6 PRTArtificial Sequence Description of Artificial Sequence synthetic peptidesequence 19 Xaa Lys Lys Lys Lys Xaa 1 5 20 6 PRT Artificial SequenceDescription of Artificial Sequence synthetic peptide sequence 20 Lys XaaXaa Lys Lys Lys 1 5 21 6 PRT Artificial Sequence Description ofArtificial Sequence synthetic peptide sequence 21 Lys Xaa Lys Xaa LysLys 1 5 22 6 PRT Artificial Sequence Description of Artificial Sequencesynthetic peptide sequence 22 Lys Xaa Lys Lys Xaa Lys 1 5 23 6 PRTArtificial Sequence Description of Artificial Sequence synthetic peptidesequence 23 Lys Xaa Lys Lys Lys Xaa 1 5 24 6 PRT Artificial SequenceDescription of Artificial Sequence synthetic peptide sequence 24 Lys LysXaa Xaa Lys Lys 1 5 25 6 PRT Artificial Sequence Description ofArtificial Sequence synthetic peptide sequence 25 Lys Lys Xaa Lys XaaLys 1 5 26 6 PRT Artificial Sequence Description of Artificial Sequencesynthetic peptide sequence 26 Lys Lys Xaa Lys Lys Xaa 1 5 27 6 PRTArtificial Sequence Description of Artificial Sequence synthetic peptidesequence 27 Lys Lys Lys Xaa Xaa Lys 1 5 28 6 PRT Artificial SequenceDescription of Artificial Sequence synthetic peptide sequence 28 Lys LysLys Xaa Lys Xaa 1 5 29 6 PRT Artificial Sequence Description ofArtificial Sequence synthetic peptide sequence 29 Lys Lys Lys Lys XaaXaa 1 5 30 7 PRT Artificial Sequence Description of Artificial Sequencesynthetic peptide sequence 30 Lys Lys Lys Lys Lys Lys Lys 1 5 31 7 PRTArtificial Sequence Description of Artificial Sequence synthetic peptidesequence 31 Xaa Lys Lys Lys Lys Lys Lys 1 5 32 7 PRT Artificial SequenceDescription of Artificial Sequence synthetic peptide sequence 32 Lys XaaLys Lys Lys Lys Lys 1 5 33 7 PRT Artificial Sequence Description ofArtificial Sequence synthetic peptide sequence 33 Lys Lys Xaa Lys LysLys Lys 1 5 34 7 PRT Artificial Sequence Description of ArtificialSequence synthetic peptide sequence 34 Lys Lys Lys Xaa Lys Lys Lys 1 535 7 PRT Artificial Sequence Description of Artificial Sequencesynthetic peptide sequence 35 Lys Lys Lys Lys Xaa Lys Lys 1 5 36 7 PRTArtificial Sequence Description of Artificial Sequence synthetic peptidesequence 36 Lys Lys Lys Lys Lys Xaa Lys 1 5 37 7 PRT Artificial SequenceDescription of Artificial Sequence synthetic peptide sequence 37 Lys LysLys Lys Lys Lys Xaa 1 5 38 7 PRT Artificial Sequence Description ofArtificial Sequence synthetic peptide sequence 38 Xaa Xaa Lys Lys LysLys Lys 1 5 39 7 PRT Artificial Sequence Description of ArtificialSequence synthetic peptide sequence 39 Xaa Lys Xaa Lys Lys Lys Lys 1 540 7 PRT Artificial Sequence Description of Artificial Sequencesynthetic peptide sequence 40 Xaa Lys Lys Xaa Lys Lys Lys 1 5 41 7 PRTArtificial Sequence Description of Artificial Sequence synthetic peptidesequence 41 Xaa Lys Lys Lys Xaa Lys Lys 1 5 42 7 PRT Artificial SequenceDescription of Artificial Sequence synthetic peptide sequence 42 Xaa LysLys Lys Lys Xaa Lys 1 5 43 7 PRT Artificial Sequence Description ofArtificial Sequence synthetic peptide sequence 43 Lys Xaa Xaa Lys LysLys Lys 1 5 44 7 PRT Artificial Sequence Description of ArtificialSequence synthetic peptide sequence 44 Lys Xaa Lys Xaa Lys Lys Lys 1 545 7 PRT Artificial Sequence Description of Artificial Sequencesynthetic peptide sequence 45 Lys Xaa Lys Lys Xaa Lys Lys 1 5 46 7 PRTArtificial Sequence Description of Artificial Sequence synthetic peptidesequence 46 Lys Xaa Lys Lys Lys Xaa Lys 1 5 47 7 PRT Artificial SequenceDescription of Artificial Sequence synthetic peptide sequence 47 Lys LysXaa Xaa Lys Lys Lys 1 5 48 7 PRT Artificial Sequence Description ofArtificial Sequence synthetic peptide sequence 48 Lys Lys Xaa Lys XaaLys Lys 1 5 49 7 PRT Artificial Sequence Description of ArtificialSequence synthetic peptide sequence 49 Lys Lys Xaa Lys Lys Xaa Lys 1 550 7 PRT Artificial Sequence Description of Artificial Sequencesynthetic peptide sequence 50 Lys Lys Lys Xaa Xaa Lys Lys 1 5 51 7 PRTArtificial Sequence Description of Artificial Sequence synthetic peptidesequence 51 Lys Lys Lys Xaa Lys Xaa Lys 1 5 52 7 PRT Artificial SequenceDescription of Artificial Sequence synthetic peptide sequence 52 Lys LysLys Lys Xaa Xaa Lys 1 5 53 7 PRT Artificial Sequence Description ofArtificial Sequence synthetic peptide sequence 53 Xaa Xaa Xaa Lys LysLys Lys 1 5 54 7 PRT Artificial Sequence Description of ArtificialSequence synthetic peptide sequence 54 Xaa Xaa Lys Xaa Lys Lys Lys 1 555 7 PRT Artificial Sequence Description of Artificial Sequencesynthetic peptide sequence 55 Xaa Xaa Lys Lys Xaa Lys Lys 1 5 56 7 PRTArtificial Sequence Description of Artificial Sequence synthetic peptidesequence 56 Xaa Xaa Lys Lys Lys Xaa Lys 1 5 57 7 PRT Artificial SequenceDescription of Artificial Sequence synthetic peptide sequence 57 Xaa LysXaa Xaa Lys Lys Lys 1 5 58 7 PRT Artificial Sequence Description ofArtificial Sequence synthetic peptide sequence 58 Xaa Lys Xaa Lys XaaLys Lys 1 5 59 7 PRT Artificial Sequence Description of ArtificialSequence synthetic peptide sequence 59 Xaa Lys Xaa Lys Lys Xaa Lys 1 560 7 PRT Artificial Sequence Description of Artificial Sequencesynthetic peptide sequence 60 Xaa Lys Lys Xaa Xaa Lys Lys 1 5 61 7 PRTArtificial Sequence Description of Artificial Sequence synthetic peptidesequence 61 Xaa Lys Lys Xaa Lys Xaa Lys 1 5 62 7 PRT Artificial SequenceDescription of Artificial Sequence synthetic peptide sequence 62 Xaa LysLys Lys Xaa Lys Xaa 1 5 63 7 PRT Artificial Sequence Description ofArtificial Sequence synthetic peptide sequence 63 Xaa Lys Lys Xaa LysLys Xaa 1 5 64 7 PRT Artificial Sequence Description of ArtificialSequence synthetic peptide sequence 64 Xaa Lys Xaa Lys Lys Lys Xaa 1 565 7 PRT Artificial Sequence Description of Artificial Sequencesynthetic peptide sequence 65 Xaa Lys Lys Lys Xaa Xaa Lys 1 5 66 7 PRTArtificial Sequence Description of Artificial Sequence synthetic peptidesequence 66 Lys Xaa Lys Lys Lys Xaa Xaa 1 5 67 7 PRT Artificial SequenceDescription of Artificial Sequence synthetic peptide sequence 67 Xaa LysLys Lys Lys Xaa Xaa 1 5 68 7 PRT Artificial Sequence Description ofArtificial Sequence synthetic peptide sequence 68 Xaa Lys Lys Lys XaaLys Xaa 1 5 69 7 PRT Artificial Sequence Description of ArtificialSequence synthetic peptide sequence 69 Xaa Lys Lys Lys Xaa Xaa Lys 1 570 7 PRT Artificial Sequence Description of Artificial Sequencesynthetic peptide sequence 70 Lys Lys Lys Lys Xaa Xaa Xaa 1 5 71 7 PRTArtificial Sequence Description of Artificial Sequence synthetic peptidesequence 71 Lys Lys Lys Xaa Xaa Xaa Lys 1 5 72 7 PRT Artificial SequenceDescription of Artificial Sequence synthetic peptide sequence 72 Lys LysLys Xaa Lys Xaa Xaa 1 5 73 7 PRT Artificial Sequence Description ofArtificial Sequence synthetic peptide sequence 73 Lys Lys Xaa Lys LysXaa Xaa 1 5 74 7 PRT Artificial Sequence Description of ArtificialSequence synthetic peptide sequence 74 Lys Lys Xaa Xaa Lys Xaa Lys 1 575 7 PRT Artificial Sequence Description of Artificial Sequencesynthetic peptide sequence 75 Lys Lys Xaa Xaa Xaa Lys Lys 1 5 76 7 PRTArtificial Sequence Description of Artificial Sequence synthetic peptidesequence 76 Lys Lys Xaa Lys Lys Xaa Xaa 1 5 77 7 PRT Artificial SequenceDescription of Artificial Sequence synthetic peptide sequence 77 Lys XaaLys Lys Xaa Xaa Lys 1 5 78 7 PRT Artificial Sequence Description ofArtificial Sequence synthetic peptide sequence 78 Lys Xaa Lys Xaa LysXaa Lys 1 5 79 7 PRT Artificial Sequence Description of ArtificialSequence synthetic peptide sequence 79 Lys Xaa Lys Xaa Xaa Lys Lys 1 580 7 PRT Artificial Sequence Description of Artificial Sequencesynthetic peptide sequence 80 Lys Xaa Xaa Lys Lys Xaa Lys 1 5 81 7 PRTArtificial Sequence Description of Artificial Sequence synthetic peptidesequence 81 Lys Xaa Xaa Lys Xaa Lys Lys 1 5 82 7 PRT Artificial SequenceDescription of Artificial Sequence synthetic peptide sequence 82 Lys XaaXaa Xaa Lys Lys Lys 1 5 83 6 PRT Artificial Sequence Description ofArtificial Sequence synthetic peptide sequence 83 Lys Glu Lys Glu LysGlu 1 5 84 6 PRT Artificial Sequence Description of Artificial Sequencesynthetic peptide sequence 84 Glu Lys Glu Lys Glu Lys 1 5 85 6 PRTArtificial Sequence Description of Artificial Sequence synthetic peptidesequence 85 Lys Lys Lys Glu Glu Glu 1 5 86 6 PRT Artificial SequenceDescription of Artificial Sequence synthetic peptide sequence 86 Glu GluGlu Lys Lys Lys 1 5 87 30 PRT Artificial Sequence Description ofArtificial Sequence Gly8-GLP-1-(7-36)(Human)-NH2 87 His Gly Glu Gly ThrPhe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15 Gln Ala Ala LysGlu Phe Ile Ala Trp Leu Val Lys Gly Arg 20 25 30 88 36 PRT ArtificialSequence Description of Artificial SequenceGly8-GLP-1-(7-36)(Human)-Lys6-NH2 88 His Gly Glu Gly Thr Phe Thr Ser AspVal Ser Ser Tyr Leu Glu Gly 1 5 10 15 Gln Ala Ala Lys Glu Phe Ile AlaTrp Leu Val Lys Gly Arg Lys Lys 20 25 30 Lys Lys Lys Lys 35 89 38 PRTArtificial Sequence Description of Artificial SequenceGly8Lys37(palmitoyl)-GLP-1-(7-36)(Human)-Lys7-NH2 89 His Gly Glu Gly ThrPhe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15 Gln Ala Ala LysGlu Phe Ile Ala Trp Leu Val Lys Gly Arg Lys Lys 20 25 30 Lys Lys Lys LysLys Lys 35 90 36 PRT Artificial Sequence Description of ArtificialSequence Gly8Lys34(palmitoyl)-GLP-1-(7-36)(Human)-Lys6-NH2 90 His GlyGlu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15 GlnAla Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Lys Lys 20 25 30 LysLys Lys Lys 35 91 44 PRT Artificial Sequence Description of ArtificialSequence des Ser39-exendin-4-Lys6-NH2 91 His Gly Glu Gly Thr Phe Thr SerAsp Leu Ser Lys Gln Met Glu Glu 1 5 10 15 Glu Ala Val Arg Leu Phe IleGlu Trp Leu Lys Asn Gly Gly Pro Ser 20 25 30 Ser Gly Ala Pro Pro Pro LysLys Lys Lys Lys Lys 35 40 92 45 PRT Artificial Sequence Description ofArtificial Sequence exendin-4-Lys6-NH2 92 His Gly Glu Gly Thr Phe ThrSer Asp Leu Ser Lys Gln Met Glu Glu 1 5 10 15 Glu Ala Val Arg Leu PheIle Glu Trp Leu Lys Asn Gly Gly Pro Ser 20 25 30 Ser Gly Ala Pro Pro ProSer Lys Lys Lys Lys Lys Lys 35 40 45 93 44 PRT Artificial SequenceDescription of Artificial Sequence des Pro36-exendin-4-Lys6-NH2 93 HisGly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu 1 5 10 15Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser 20 25 30Ser Gly Ala Pro Pro Ser Lys Lys Lys Lys Lys Lys 35 40 94 44 PRTArtificial Sequence Description of Artificial Sequence desAla35-exendin-4-Lys6-NH2 94 His Gly Glu Gly Thr Phe Thr Ser Asp Leu SerLys Gln Met Glu Glu 1 5 10 15 Glu Ala Val Arg Leu Phe Ile Glu Trp LeuLys Asn Gly Gly Pro Ser 20 25 30 Ser Gly Pro Pro Pro Ser Lys Lys Lys LysLys Lys 35 40 95 44 PRT Artificial Sequence Description of ArtificialSequence des Gly34-exendin-4-Lys6-NH2 95 His Gly Glu Gly Thr Phe Thr SerAsp Leu Ser Lys Gln Met Glu Glu 1 5 10 15 Glu Ala Val Arg Leu Phe IleGlu Trp Leu Lys Asn Gly Gly Pro Ser 20 25 30 Ser Ala Pro Pro Pro Ser LysLys Lys Lys Lys Lys 35 40 96 46 PRT Artificial Sequence Description ofArtificial Sequence des Ser39-(Lys40(palmitoyl))exendin-4-Lys7-NH2 96His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu 1 5 1015 Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser 20 2530 Ser Gly Ala Pro Pro Pro Lys Lys Lys Lys Lys Lys Lys Lys 35 40 45 9746 PRT Artificial Sequence Description of Artificial Sequence desGly34-(Lys40(palmitoyl))exendin-4-Lys7-NH2 97 His Gly Glu Gly Thr PheThr Ser Asp Leu Ser Lys Gln Met Glu Glu 1 5 10 15 Glu Ala Val Arg LeuPhe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser 20 25 30 Ser Ala Pro Pro ProSer Lys Lys Lys Lys Lys Lys Lys Lys 35 40 45 98 46 PRT ArtificialSequence Description of Artificial Sequence desAla35-(Lys40(palmitoyl))exendin-4-Lys7-NH2 98 His Gly Glu Gly Thr PheThr Ser Asp Leu Ser Lys Gln Met Glu Glu 1 5 10 15 Glu Ala Val Arg LeuPhe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser 20 25 30 Ser Gly Pro Pro ProSer Lys Lys Lys Lys Lys Lys Lys Lys 35 40 45 99 46 PRT ArtificialSequence Description of Artificial Sequence desPro36-(Lys40(palmitoyl))exendin-4-Lys7-NH2 99 His Gly Glu Gly Thr PheThr Ser Asp Leu Ser Lys Gln Met Glu Glu 1 5 10 15 Glu Ala Val Arg LeuPhe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser 20 25 30 Ser Gly Ala Pro ProSer Lys Lys Lys Lys Lys Lys Lys Lys 35 40 45 100 47 PRT ArtificialSequence Description of Artificial SequenceLys40(palmitoyl)exendin-4-Lys7-NH2 100 His Gly Glu Gly Thr Phe Thr SerAsp Leu Ser Lys Gln Met Glu Glu 1 5 10 15 Glu Ala Val Arg Leu Phe IleGlu Trp Leu Lys Asn Gly Gly Pro Ser 20 25 30 Ser Gly Ala Pro Pro Pro SerLys Lys Lys Lys Lys Lys Lys Lys 35 40 45 101 38 PRT Artificial SequenceDescription of Artificial Sequence des Pro36-exendin-4-NH2 101 His GlyGlu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu 1 5 10 15 GluAla Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser 20 25 30 SerGly Ala Pro Pro Ser 35 102 39 PRT Homo sapiens exendin-4-NH2 102 His GlyGlu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu 1 5 10 15 GluAla Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser 20 25 30 SerGly Ala Pro Pro Pro Ser 35 103 36 PRT Artificial Sequence Description ofArtificial Sequence Gly8Lys26(palmitoyl)-GLP-1-(7-36)(Human)-Lys6-NH2103 His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 510 15 Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Lys Lys 2025 30 Lys Lys Lys Lys 35 104 38 PRT Artificial Sequence Description ofArtificial Sequence des Ser39-exendin-4-NH2 104 His Gly Glu Gly Thr PheThr Ser Asp Leu Ser Lys Gln Met Glu Glu 1 5 10 15 Glu Ala Val Arg LeuPhe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser 20 25 30 Ser Gly Ala Pro ProPro 35 105 38 PRT Artificial Sequence Description of Artificial Sequencedes Ala35-exendin-4-NH2 105 His Gly Glu Gly Thr Phe Thr Ser Asp Leu SerLys Gln Met Glu Glu 1 5 10 15 Glu Ala Val Arg Leu Phe Ile Glu Trp LeuLys Asn Gly Gly Pro Ser 20 25 30 Ser Gly Pro Pro Pro Ser 35 106 38 PRTArtificial Sequence Description of Artificial Sequence desGly34-exendin-4-NH2 106 His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser LysGln Met Glu Glu 1 5 10 15 Glu Ala Val Arg Leu Phe Ile Glu Trp Leu LysAsn Gly Gly Pro Ser 20 25 30 Ser Ala Pro Pro Pro Ser 35 107 39 PRTArtificial Sequence Description of Artificial Sequence des Ser39-(Lys40(palmitoyl))exendin-4-NH2 107 His Gly Glu Gly Thr Phe Thr Ser Asp LeuSer Lys Gln Met Glu Glu 1 5 10 15 Glu Ala Val Arg Leu Phe Ile Glu TrpLeu Lys Asn Gly Gly Pro Ser 20 25 30 Ser Gly Ala Pro Pro Pro Lys 35 10839 PRT Artificial Sequence Description of Artificial Sequence desGly34-(Lys40 (palmitoyl))exendin-4-NH2 108 His Gly Glu Gly Thr Phe ThrSer Asp Leu Ser Lys Gln Met Glu Glu 1 5 10 15 Glu Ala Val Arg Leu PheIle Glu Trp Leu Lys Asn Gly Gly Pro Ser 20 25 30 Ser Ala Pro Pro Pro SerLys 35 109 39 PRT Artificial Sequence Description of Artificial Sequencedes Ala35-(Lys40 (palmitoyl))exendin-4-NH2 109 His Gly Glu Gly Thr PheThr Ser Asp Leu Ser Lys Gln Met Glu Glu 1 5 10 15 Glu Ala Val Arg LeuPhe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser 20 25 30 Ser Gly Pro Pro ProSer Lys 35 110 39 PRT Artificial Sequence Description of ArtificialSequence des Pro36-(Lys40 (palmitoyl))exendin-4-NH2 110 His Gly Glu GlyThr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu 1 5 10 15 Glu Ala ValArg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser 20 25 30 Ser Gly AlaPro Pro Ser Lys 35 111 31 PRT Artificial Sequence Description ofArtificial Sequence Gly8Lys37N-palmitoyl-GLP-1 (7-36) 111 His Gly GluGly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15 Gln AlaAla Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Lys 20 25 30 112 30 PRTArtificial Sequence Description of Artificial SequenceGly8Lys34N-palmitoyl-GLP-1 (7-36) 112 His Gly Glu Gly Thr Phe Thr SerAsp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15 Gln Ala Ala Lys Glu Phe IleAla Trp Leu Val Lys Gly Arg 20 25 30 113 30 PRT Artificial SequenceDescription of Artificial Sequence Gly8Lys26N-palmitoyl-GLP-1 (7-36) 113His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 1015 Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg 20 25 30 11430 PRT Homo sapiens GLP-1(7-36) 114 His Ala Glu Gly Thr Phe Thr Ser AspVal Ser Ser Tyr Leu Glu Gly 1 5 10 15 Gln Ala Ala Lys Glu Phe Ile AlaTrp Leu Val Lys Gly Arg 20 25 30 115 36 PRT Artificial SequenceDescription of Artificial Sequence Ser8-GLP-1(7-36)-Lys6 115 His Ser GluGly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15 Gln AlaAla Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Lys Lys 20 25 30 Lys LysLys Lys 35 116 36 PRT Artificial Sequence MOD_RES (2) Aib 116 His XaaGlu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15 GlnAla Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Lys Lys 20 25 30 LysLys Lys Lys 35 117 37 PRT Artificial Sequence Description of ArtificialSequence Gly8-GLP-1(7-36)-Lys7 117 His Gly Glu Gly Thr Phe Thr Ser AspVal Ser Ser Tyr Leu Glu Gly 1 5 10 15 Gln Ala Ala Lys Glu Phe Ile AlaTrp Leu Val Lys Gly Arg Lys Lys 20 25 30 Lys Lys Lys Lys Lys 35 118 42PRT Artificial Sequence Description of Artificial SequenceLys6-Gly8-GLP-1(7-36)-Lys6 118 Lys Lys Lys Lys Lys Lys His Gly Glu GlyThr Phe Thr Ser Asp Val 1 5 10 15 Ser Ser Tyr Leu Glu Gly Gln Ala AlaLys Glu Phe Ile Ala Trp Leu 20 25 30 Val Lys Gly Arg Lys Lys Lys Lys LysLys 35 40 119 36 PRT Artificial Sequence Description of ArtificialSequence Lys6-Gly8-GLP-1(7-36) 119 Lys Lys Lys Lys Lys Lys His Gly GluGly Thr Phe Thr Ser Asp Val 1 5 10 15 Ser Ser Tyr Leu Glu Gly Gln AlaAla Lys Glu Phe Ile Ala Trp Leu 20 25 30 Val Lys Gly Arg 35 120 38 PRTArtificial Sequence Description of Artificial SequenceGly8-GLP-1(7-36)-Lys8 120 His Gly Glu Gly Thr Phe Thr Ser Asp Val SerSer Tyr Leu Glu Gly 1 5 10 15 Gln Ala Ala Lys Glu Phe Ile Ala Trp LeuVal Lys Gly Arg Lys Lys 20 25 30 Lys Lys Lys Lys Lys Lys 35 121 40 PRTArtificial Sequence Description of Artificial SequenceGly8-GLP-1(7-36)-Lys10 121 His Gly Glu Gly Thr Phe Thr Ser Asp Val SerSer Tyr Leu Glu Gly 1 5 10 15 Gln Ala Ala Lys Glu Phe Ile Ala Trp LeuVal Lys Gly Arg Lys Lys 20 25 30 Lys Lys Lys Lys Lys Lys Lys Lys 35 40122 37 PRT Artificial Sequence Description of Artificial SequenceGly8-GLP-1(7-37)-Lys6 122 His Gly Glu Gly Thr Phe Thr Ser Asp Val SerSer Tyr Leu Glu Gly 1 5 10 15 Gln Ala Ala Lys Glu Phe Ile Ala Trp LeuVal Lys Gly Arg Gly Lys 20 25 30 Lys Lys Lys Lys Lys 35 123 31 PRTArtificial Sequence Description of Artificial Sequence Gly8-GLP-1(7-37)123 His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 510 15 Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly 20 2530 124 31 PRT Homo sapiens GLP-1(7-37) 124 His Ala Glu Gly Thr Phe ThrSer Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15 Gln Ala Ala Lys Glu PheIle Ala Trp Leu Val Lys Gly Arg Gly 20 25 30 125 28 PRT ArtificialSequence Description of Artificial Sequence GLP-1(9-36)(Human) 125 GluGly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala 1 5 10 15Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg 20 25 126 39 PRTArtificial Sequence Description of Artificial Sequence [Tyr39]exendin-4126 His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu 1 510 15 Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser 2025 30 Ser Gly Ala Pro Pro Pro Tyr 35 127 30 PRT Homo sapiens exendin-4(1-31) 127 His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met GluGlu 1 5 10 15 Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly 2025 30 128 30 PRT Homo sapiens exendin4 (9-39) 128 Leu Ser Lys Gln MetGlu Glu Glu Ala Val Arg Leu Phe Ile Glu Trp 1 5 10 15 Leu Lys Asn GlyGly Pro Ser Ser Gly Ala Pro Pro Pro Ser 20 25 30 129 38 PRT ArtificialSequence Description of Artificial Sequence des Ser39-exendin-4 129 HisGly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu 1 5 10 15Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser 20 25 30Ser Gly Ala Pro Pro Pro 35 130 37 PRT Artificial Sequence Description ofArtificial Sequence des-Pro36,Pro37-exendin-4 130 His Gly Glu Gly ThrPhe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu 1 5 10 15 Glu Ala Val ArgLeu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser 20 25 30 Ser Gly Ala ProSer 35 131 43 PRT Artificial Sequence Description of Artificial Sequencedes-Pro36,Pro37-exendin-4-Lys6 131 His Gly Glu Gly Thr Phe Thr Ser AspLeu Ser Lys Gln Met Glu Glu 1 5 10 15 Glu Ala Val Arg Leu Phe Ile GluTrp Leu Lys Asn Gly Gly Pro Ser 20 25 30 Ser Gly Ala Pro Ser Lys Lys LysLys Lys Lys 35 40 132 36 PRT Artificial Sequence Description ofArtificial Sequence des-Pro36,Pro37, Pro38-exendin-4 132 His Gly Glu GlyThr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu 1 5 10 15 Glu Ala ValArg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser 20 25 30 Ser Gly AlaSer 35 133 42 PRT Artificial Sequence Description of Artificial Sequencedes-Pro36,Pro37, Pro38-exendin-4-Lys6 133 His Gly Glu Gly Thr Phe ThrSer Asp Leu Ser Lys Gln Met Glu Glu 1 5 10 15 Glu Ala Val Arg Leu PheIle Glu Trp Leu Lys Asn Gly Gly Pro Ser 20 25 30 Ser Gly Ala Ser Lys LysLys Lys Lys Lys 35 40 134 42 PRT Artificial Sequence Description ofArtificial Sequence Lys6-des-Pro36 ,Pro37, Pro38-exendin-4 134 Lys LysLys Lys Lys Lys His Gly Glu Gly Thr Phe Thr Ser Asp Leu 1 5 10 15 SerLys Gln Met Glu Glu Glu Ala Val Arg Leu Phe Ile Glu Trp Leu 20 25 30 LysAsn Gly Gly Pro Ser Ser Gly Ala Ser 35 40 135 48 PRT Artificial SequenceDescription of Artificial Sequence Lys6-des-Pro36 ,Pro37,Pro38-exendin-4-Lys6 135 Lys Lys Lys Lys Lys Lys His Gly Glu Gly Thr PheThr Ser Asp Leu 1 5 10 15 Ser Lys Gln Met Glu Glu Glu Ala Val Arg LeuPhe Ile Glu Trp Leu 20 25 30 Lys Asn Gly Gly Pro Ser Ser Gly Ala Ser LysLys Lys Lys Lys Lys 35 40 45 136 48 PRT Artificial Sequence Descriptionof Artificial Sequence Asn(Glu)5-des-Pro36,Pro37, Pro38-exendin-4-Lys6136 Asn Glu Glu Glu Glu Glu His Gly Glu Gly Thr Phe Thr Ser Asp Leu 1 510 15 Ser Lys Gln Met Glu Glu Glu Ala Val Arg Leu Phe Ile Glu Trp Leu 2025 30 Lys Asn Gly Gly Pro Ser Ser Gly Ala Ser Lys Lys Lys Lys Lys Lys 3540 45 137 42 PRT Artificial Sequence Description of Artificial SequenceAsn(Glu)5-des-Pro36,Pro37, Pro38-exendin-4 137 Asn Glu Glu Glu Glu GluHis Gly Glu Gly Thr Phe Thr Ser Asp Leu 1 5 10 15 Ser Lys Gln Met GluGlu Glu Ala Val Arg Leu Phe Ile Glu Trp Leu 20 25 30 Lys Asn Gly Gly ProSer Ser Gly Ala Ser 35 40 138 40 PRT Homo sapiens Exendin-4 (1-40) 138His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu 1 5 1015 Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser 20 2530 Ser Gly Ala Pro Pro Pro Ser Gly 35 40 139 39 PRT Artificial SequenceDescription of Artificial Sequence des-Pro36-exendin-4 (1-40) 139 HisGly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu 1 5 10 15Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser 20 25 30Ser Gly Ala Pro Pro Ser Gly 35 140 37 PRT Artificial SequenceDescription of Artificial Sequence des-Pro36,Pro37, Pro38-exendin-4(1-40) 140 His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met GluGlu 1 5 10 15 Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly GlyPro Ser 20 25 30 Ser Gly Ala Ser Gly 35 141 6 PRT Artificial SequenceDescription of Artificial Sequence Synthetic Peptide Sequence 141 AsnGlu Glu Glu Glu Glu 1 5 142 7 PRT Artificial Sequence Description ofArtificial Sequence Synthetic Peptide Sequence 142 Asn Glu Glu Glu GluGlu Glu 1 5 143 6 PRT Artificial Sequence Description of ArtificialSequence Synthetic Peptide Sequence 143 Gln Glu Glu Glu Glu Glu 1 5 1446 PRT Artificial Sequence Description of Artificial Sequence SyntheticPeptide Sequence 144 Asn Asp Asp Asp Asp Asp 1 5 145 6 PRT ArtificialSequence Description of Artificial Sequence Synthetic Peptide Sequence145 Gln Asp Asp Asp Asp Asp 1 5 146 138 DNA Artificial SequenceDescription of Artificial Sequence Synthetic cDNA 146 atgcatggtgagggtacatt cacatctgat ttgtctaagc aaatggagga ggaggctgtt 60 cgtttgttcattgagtggtt gaagaatggt ggtccatctt ctggtgctcc accatctaag 120 aagaagaagaagaagtaa 138 147 32 PRT Artificial Sequence MOD_RES (32) Lys(palmitoyl)147 His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 510 15 Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Lys 2025 30 148 31 PRT Artificial Sequence Description of Artificial SequenceY31-exendin-4 (1-31)(Human) 148 His Gly Glu Gly Thr Phe Thr Ser Asp LeuSer Lys Gln Met Glu Glu 1 5 10 15 Glu Ala Val Arg Leu Phe Ile Glu TrpLeu Lys Asn Gly Gly Lys 20 25 30 149 44 PRT Artificial SequenceDescription of Artificial Sequence Lys6-des-Pro36-exendin-4 149 Lys LysLys Lys Lys Lys His Gly Glu Gly Thr Phe Thr Ser Asp Leu 1 5 10 15 SerLys Gln Met Glu Glu Glu Ala Val Arg Leu Phe Ile Glu Trp Leu 20 25 30 LysAsn Gly Gly Pro Ser Ser Gly Ala Pro Pro Ser 35 40 150 50 PRT ArtificialSequence Description of Artificial SequenceLys6-des-Pro36-exendin-4-Lys6 150 Lys Lys Lys Lys Lys Lys His Gly GluGly Thr Phe Thr Ser Asp Leu 1 5 10 15 Ser Lys Gln Met Glu Glu Glu AlaVal Arg Leu Phe Ile Glu Trp Leu 20 25 30 Lys Asn Gly Gly Pro Ser Ser GlyAla Pro Pro Ser Lys Lys Lys Lys 35 40 45 Lys Lys 50 151 40 PRTArtificial Sequence MOD_RES (40) Lys(palmitoyl) 151 His Gly Glu Gly ThrPhe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu 1 5 10 15 Glu Ala Val ArgLeu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser 20 25 30 Ser Gly Ala ProPro Pro Ser Lys 35 40 152 39 PRT Artificial Sequence MOD_RES (39)Lys(palmitoyl) 152 His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys GlnMet Glu Glu 1 5 10 15 Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys AsnGly Gly Pro Ser 20 25 30 Ser Gly Ala Pro Pro Ser Lys 35 153 37 PRTArtificial Sequence Description of Artificial SequenceGly8Lys37(palmitoyl)-Glp-1-(7-36)(Human)-Lys6 153 His Gly Glu Gly ThrPhe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15 Gln Ala Ala LysGlu Phe Ile Ala Trp Leu Val Lys Gly Arg Lys Lys 20 25 30 Lys Lys Lys LysLys 35

1. A peptide conjugate comprising a peptide X selected from the groupconsisting of (a) an exendin having at least 90% homology to exendin-4;(b) a variant of said exendin wherein said variant comprises amodification selected from the group consisting of between one and fivedeletions at positions 34-39 and contains a Lys at position 40 having alipophilic substituent; or (c) GLP-1 (7-36) (SEQ ID NO: 114) or GLP-1(7-37) (SEQ ID NO: 123) having at least one modification selected fromthe group consisting of: (i) substitution of D-alanine, glycine oralpha-amino isobutyric acid for alanine at position 8 and (ii) alipophilic substituent; and Z, a peptide sequence of 4-20.amino acidunits covalently bound to said variant, wherein each amino acid unit insaid peptide sequence, Z is selected from the group consisting of Ala,Leu, Ser, Thr, Tyr, Asn, Gin, Asp, Glu, Lys, Arg, His, Met, Orn, andamino acid units of the general formula I —NH—C(R¹)(R²)—C(═O)—(1)wherein R¹ and R² are selected from the group consisting of hydrogen,C₁₋₆-alkyl, phenyl, and phenyl-methyl, wherein C₁₋₆-alkyl is optionallysubstituted with from one to three substituents selected from halogen,hydroxy, amino, cyano, nitro, sulfono, and carboxy, and phenyl andphenyl-methyl is optionally substituted with from one to threesubstituents selected from C₁₋₆-alkyl, C₂₋₆-alkenyl, halogen, hydroxy,amino, cyano, nitro, sulfono, and carboxy, or R¹ and R² together withthe carbon atom to which they are bound form a cyclopentyl, cyclohexyl,or cycloheptyl ring, e.g. 2,4-diaminobutanoic acid and2,3-diaminopropanoic acid; and a pharmaceutically acceptable salt or thec-terminal amide of said peptide conjugate, with the proviso that x isnot exendin-4 or exendin-3.
 2. The peptide conjugate according to claim1, wherein the peptide X is further characterised in being effective inimproving glucose tolerance in a diabetic mammal.
 3. The peptideconjugate according to claim 1 or 2, wherein Z comprises at least oneLys amino acid unit, preferably at least two Lys amino acid units, suchas at least three Lys amino acid units, e.g. at least four Lys aminoacid units, more preferably at least five Lys amino acid units, such assix or seven Lys amino acid units.
 4. A peptide conjugate according toany one of the preceding claims, characterised in that the ratio betweenthe minimum effective dose of said peptide conjugate and the minimumeffective oral dose of the peptide, X is at least 1:5.
 5. The peptideconjugate according to any one of the preceding claims, wherein thelipophilic substituent is a palmitoyl attached to the epsilon aminogroup of a Lys in peptide X.
 6. The peptide conjugate according to anyone of the preceding claims, wherein X is selected from the groupconsisting of Gly⁸-GLP-1-(7-36)(Human)-NH₂(SEQ ID NO: 87),Gly⁸-GLP-1-(7-36)(Human) (SEQ ID NO: 87),Gly⁸Lys³⁷(palmitoyl)-GLP-1-(7-36)(Human) (SEQ ID NO: 111),Gly⁸Lys³⁴(palmitoyl-GLP-1-(7-36)(Human) (SEQ ID NO: 90), desSer³⁹-exendin-4(1-39) (SEQ ID NO: 129), exendin-4(1-39) (SEQ ID NO:102), des Pro-exendin-4(1-39) (SEQ ID NO: 101), desAla³⁵-exendin-4(1-39) (SEQ ID NO: 105), des Gly³⁴-exendin-4(1-39) (SEQID NO: 106), des Ser³⁹-(Lys⁴⁰ (palmitoyl))exendin-4(1-39) (SEQ ID NO:107), des Gly³⁴-(Lys (palmitoyl))exendin-4(1-39) (SEQ ID NO: 108), desAla³⁵-(Lys⁴⁰ (palmitoyl))exendin-4(1-39) (SEQ ID NO: 109), desPro³⁶-(Lys⁴⁰(palmitoyl))exendin-4(1-39) (SEQ ID NO: 110) and Lys⁴⁰(palmitoyl)exendin-4(1-39) (SEQ ID NO: 151), and the free acids orC-terminal primary amides and pharmaceutically acceptable salts thereof.7. A peptide conjugate according to any one of claims 1-5, wherein saidpeptide X is selected from the group consisting of a GLP-1 (7-36)-NH₂(SEQ ID NO: 114) having at least one modification selected from thegroup consisting of substitution of glycine for alanine at position 8and a lipophilic palmitoyl group attached to a lysine at position 26, 34or 37, and the free acid and a pharmaceutically acceptable salt thereof.8. The peptide conjugate according to the preceding claim, wherein thelipophilic substituent is attached to the epsilon amino group of a Lysin said GLP-1.
 9. A peptide conjugate according to any one of claims1-5, wherein said peptide X is selected from the group consisting of anexendin variant having an amino acid sequence wherein 1,2 or 3 of thePro residues in positions 36-38 have been deleted from the amino acidsequence of exendin-4, preferably X is selected from the groupconsisting of exendin variants having an amino acid sequence wherein 1,2 or 3 of the Pro residues in positions 36-38 have been deleted from theamino acid sequence of exendin-4(1-39) (SEQ ID NO: 102), the free acidor C-terminal primary amide thereof, and a pharmaceutically acceptablesalt thereof.
 10. A peptide conjugate according to the preceding claim,wherein X is selected from the group consisting of desPro³⁶-exendin-4(1-39)-NH₂(SEQ ID NO: 101), des Pro³⁶,Pro³⁷-exendin-4(1-39)-NH₂(SEQ ID NO: 130), and des Pro³⁶, Pro³⁷,Pro³⁸-exendin-4(1-39)-NH₂(SEQ ID NO: 132).
 11. A peptide conjugateaccording to any one of the preceding claims, characterised in that X isbound to Z via a peptide bond.
 12. The peptide conjugate according toclaim 1, wherein Z is covalently bound to X at the C-terminal carbonylfunction of X, or Z is covalently bound to the N-terminal nitrogen atomof said variant, or the first sequence (Z) is covalently bound to X atthe C-terminal carbonyl function of said variant and the second sequence(Z) is covalently bound to the N-terminal nitrogen atom of X, or Z iscovalently bound to a nitrogen atom on the side chain of a lysine,arginine or histidine residue or a carbonyl function on the side chainof glutamic acid or aspartic acid of said variant.
 13. The peptideconjugate according to any one of the preceding claims, wherein Zconsists of 4-15, preferably 4-10, more preferably 4-7, most preferably6-7 amino acid units.
 14. A peptide conjugate according to any one ofthe preceding claims, wherein the overall charge of the peptide sequence(Z) is in the range from 0 to +15 at pH 7, preferably in the range from0 to +10, such as in the range from 0 to +8, more preferably in therange from 0 to +7 or 0 to +6.
 15. The peptide conjugate according toany one of the preceding claims, wherein each amino acid unit in Z isindependently selected from the group consisting of Ser, Thr, Tyr, Asn,Gln, Asp, Glu, Lys, Arg, His, Orn, 2,4-diaminobutanoic acid,2,3-diaminopropanoic acid and Met.
 16. A peptide conjugate according toany one of the preceding claims, wherein Z is (Lys)_(n), wherein n is aninteger in the range from 4 to 15, preferably in the range from 4 to 10,such as in the range from 4 to 8, e.g. in the range from 4 to
 7. 17. Apeptide conjugate according to the preceding claim wherein Z is Lys₆(SEQ ID NO: 9) or Lys₇(SEQ ID NO: 30).
 18. A peptide conjugate accordingto claim 1 wherein Z is represented by Lys₆ bound to the C-terminal ofX.
 19. A peptide conjugate according to claim 1 wherein Z is attached toX N-terminally via a peptide bond and wherein the amino acid residuesare selected from the group consisting of Asn, Asp, Glu, and Gin,preferably Asn and Glu, preferably Z has an amino acid sequence selectedfrom the group consisting of Asn-(Glu)_(n) wherein n is an integer from3 to 7, preferably
 5. 20. A peptide conjugate according to claim 13,wherein Z is Lys-Lys-Lys-Lys (SEQ ID NO: 1), Xaa-Lys-Lys-Lys,Lys-Xaa-Lys-Lys, Lys-Lys-Xaa-Lys, Lys-Lys-Lys-Xaa, Xaa-Xaa-Lys-Lys,Xaa-Lys-Xaa-Lys, Xaa-Lys-Lys-Xaa, Lys-Xaa-Xaa-Lys, Lys-Xaa-Lys-Xaa,Lys-Lys-Xaa-Xaa, Xaa-Xaa-Xaa-Lys, Xaa-Xaa-Lys-Xaa, Xaa-Lys-Xaa-Xaa,Lys-Xaa-Xaa-Xaa, Xaa-Xaa-Xaa-Xaa (SEQ ID NO:2), Lys-Lys-Lys-Lys-Lys (SEQID NO:3), Xaa-Lys-Lys-Lys-Lys (SEQ ID NO:4), Lys-Xaa-Lys-Lys-Lys (SEQ IDNO:5), Lys-Lys-Xaa-Lys-Lys (SEQ ID NO:6), Lys-Lys-Lys-Xaa-Lys (SEQ IDNO:7), Lys-Lys-Lys-Lys-Xaa, Xaa-Xaa-Lys-Lys-Lys, Xaa-Lys-Xaa-Lys-Lys,Xaa-Lys-Lys-Xaa-Lys, Xaa-Lys-Lys-Lys-Xaa, Lys-Xaa-Xaa-Lys-Lys,Lys-Xaa-Lys-Xaa-Lys, Lys-Xaa-Lys-Lys-Xaa, Lys-Lys-Xaa-Xaa-Lys,Lys-Lys-Xaa-Lys-Xaa, Lys-Lys-Lys-Xaa-Xaa, Lys-Lys-Xaa-Xaa-Xaa,Lys-Xaa-Lys-Xaa-Xaa, Lys-Xaa-Xaa-Lys-Xaa, Lys-Xaa-Xaa-Xaa-Lys,Xaa-Lys-Lys-Xaa-Xaa, Xaa-Lys-Xaa-Xaa-Lys, Xaa-Xaa-Lys-Lys-Xaa,Xaa-Xaa-Lys-Xaa-Lys, Xaa-Xaa-Xaa-Lys-Lys, Lys-Xaa-Xaa-Xaa-Xaa,Xaa-Lys-Xaa-Xaa-Xaa, Xaa-Xaa-Lys-Xaa-Xaa, Xaa-Xaa-Xaa-Lys-Xaa,Xaa-Xaa-Xaa-Xaa-Lys, Xaa-Xaa-Xaa-Xaa-Xaa (SEQ ID NO:8),Lys-Lys-Lys-Lys-Lys-Lys (SEQ ID NO:9), Xaa-Lys-Lys-Lys-Lys-Lys (SEQ IDNO:10), Lys-Xaa-Lys-Lys-Lys-Lys (SEQ ID NO:11), Lys-Lys-Xaa-Lys-Lys-Lys(SEQ ID NO:12), Lys-Lys-Lys-Xaa-Lys-Lys (SEQ ID NO:13),Lys-Lys-Lys-Lys-Xaa-Lys (SEQ ID NO:14), Lys-Lys-Lys-Lys-Lys-Xaa (SEQ IDNO: 15), Xaa-Xaa-Lys-Lys-Lys-Lys (SEQ ID NO: 16),Xaa-Lys-Xaa-Lys-Lys-Lys (SEQ ID NO:17), Xaa-Lys-Lys-Xaa-Lys-Lys (SEQ IDNO:18), Xaa-Lys-Lys-Lys-Xaa-Lys (SEQ ID NO: 19), Xaa-Lys-Lys-Lys-Lys-Xaa(SEQ ID NO:20), Lys-Xaa-Xaa-Lys-Lys-Lys (SEQ ID NO:21),Lys-Xaa-Lys-Xaa-Lys-Lys (SEQ ID NO:22), Lys-Xaa-Lys-Lys-Xaa-Lys (SEQ IDNO:23), Lys-Xaa-Lys-Lys-Lys-Xaa (SEQ ID NO:24), Lys-Lys-Xaa-Xaa-Lys-Lys(SEQ ID NO:25), Lys-Lys-Xaa-Lys-Xaa-Lys (SEQ ID NO:26),Lys-Lys-Xaa-Lys-Lys-Xaa (SEQ ID NO:27), Lys-Lys-Lys-Xaa-Xaa-Lys (SEQ IDNO:28), Lys-Lys-Lys-Xaa-Lys-Xaa (SEQ ID NO:29), Lys-Lys-Lys-Lys-Xaa-Xaa,Xaa-Xaa-Xaa-Lys-Lys-Lys, Xaa-Xaa-Lys-Xaa-Lys-Lys,Xaa-Xaa-Lys-Lys-Xaa-Lys, Xaa-Xaa-Lys-Lys-Lys-Xaa,Xaa-Lys-Xaa-Xaa-Lys-Lys, Xaa-Lys-Xaa-Lys-Xaa-Lys,Xaa-Lys-Xaa-Lys-Lys-Xaa, Xaa-Lys-Lys-Xaa-Xaa-Lys,Xaa-Lys-Lys-Xaa-Lys-Xaa, Xaa-Lys-Lys-Lys-Xaa-Xaa,Lys-Lys-Lys-Xaa-Xaa-Xaa, Lys-Lys-Xaa-Lys-Xaa-Xaa,Lys-Lys-Xaa-Xaa-Lys-Xaa, Lys-Lys-Xaa-Xaa-Xaa-Lys,Lys-Xaa-Lys-Lys-Xaa-Xaa, Lys-Xaa-Lys-Xaa-Lys-Xaa,Lys-Xaa-Lys-Xaa-Xaa-Lys, Lys-Xaa-Xaa Lys-Lys-Xaa,Lys-Xaa-Xaa-Lys-Xaa-Lys, Lys-Xaa-Xaa-Xaa-Lys-Lys,Lys-Lys-Xaa-Xaa-Xaa-Xaa, Lys-Xaa-Lys-Xaa-Xaa-Xaa,Lys-Xaa-Xaa-Lys-Xaa-Xaa-Lys, Lys-Xaa-Xaa-Xaa-Lys-Xaa-Lys,Lys-Xaa-Xaa-Xaa-Xaa-Lys-Lys, Xaa-Lys-Lys-Xaa-Xaa-Xaa,Xaa-Lys-Xaa-Lys-Xaa-Xaa, Xaa-Lys-Xaa-Xaa-Lys-Xaa,Xaa-Lys-Xaa-Xaa-Xaa-Lys, Xaa-Xaa-Lys-Lys-Xaa-Xaa,Xaa-Xaa-Lys-Xaa-Lys-Xaa, Xaa-Xaa-Lys-Xaa-Xaa-Lys,Xaa-Xaa-Xaa-Lys-Lys-Xaa, Xaa-Xaa-Xaa-Lys-Xaa-Lys,Xaa-Xaa-Xaa-Xaa-Lys-Lys, Lys-Xaa-Xaa-Xaa-Xaa-Xaa,Xaa-Lys-Xaa-Xaa-Xaa-Xaa, Xaa-Xaa-Lys-Xaa-Xaa-Xaa,Xaa-Xaa-Xaa-Lys-Xaa-Xaa, Xaa-Xaa-Xaa-Xaa-Lys-Xaa,Xaa-Xaa-Xaa-Xaa-Xaa-Lys, Xaa-Xaa-Xaa-Xaa-Xaa-Xaa,Lys-Lys-Lys-Lys-Lys-Lys-Lys (SEQ ID NO:30), Xaa-Lys-Lys-Lys-Lys-Lys-Lys(SEQ ID NO:31), Lys-Xaa-Lys-Lys-Lys-Lys-Lys (SEQ ID NO:32),Lys-Lys-Xaa-Lys-Lys-Lys-Lys (SEQ ID NO:33), Lys-Lys-Lys-Xaa-Lys-Lys-Lys(SEQ ID NO:34), Lys-Lys-Lys-Lys-Xaa-Lys-Lys (SEQ ID NO:35),Lys-Lys-Lys-Lys-Lys-Xaa-Lys (SEQ ID NO:36), Lys-Lys-Lys-Lys-Lys-Lys-Xaa(SEQ ID NO:37), Xaa-Xaa-Lys-Lys-Lys-Lys-Lys (SEQ ID NO:38),Xaa-Lys-Xaa-Lys-Lys-Lys-Lys (SEQ ID NO:39), Xaa-Lys-Lys-Xaa-Lys-Lys-Lys(SEQ ID NO:40), Xaa-Lys-Lys-Lys-Xaa-Lys-Lys (SEQ ID NO:41),Xaa-Lys-Lys-Lys-Lys-Xaa-Lys (SEQ ID NO:42), Lys-Xaa-Xaa-Lys-Lys-Lys-Lys(SEQ ID NO:43), Lys-Xaa-Lys-Xaa-Lys-Lys-Lys (SEQ ID NO:44),Lys-Xaa-Lys-Lys-Xaa-Lys-Lys (SEQ ID NO:45), Lys-Xaa-Lys-Lys-Lys-Xaa-Lys(SEQ ID NO:46), Lys-Lys-Xaa-Xaa-Lys-Lys-Lys (SEQ ID NO:47),Lys-Lys-Xaa-Lys-Xaa-Lys-Lys (SEQ ID NO:48), Lys-Lys-Xaa-Lys-Lys-Xaa-Lys(SEQ ID NO:49), Lys-Lys-Lys-Xaa-Xaa-Lys-Lys (SEQ ID NO:50),Lys-Lys-Lys-Xaa-Lys-Xaa-Lys (SEQ ID NO:51), Lys-Lys-Lys-Lys-Xaa-Xaa-Lys(SEQ ID NO:52), Xaa-Xaa-Xaa-Lys-Lys-Lys-Lys (SEQ ID NO:53),Xaa-Xaa-Lys-Xaa-Lys-Lys-Lys (SEQ ID NO:54), Xaa-Xaa-Lys-Lys-Xaa-Lys-Lys(SEQ ID NO:55), Xaa-Xaa-Lys-Lys-Lys-Xaa-Lys (SEQ ID NO:56),Xaa-Lys-Xaa-Xaa-Lys-Lys-Lys (SEQ ID NO:57), Xaa-Lys-Xaa-Lys-Xaa-Lys-Lys(SEQ ID NO:58), Xaa-Lys-Xaa-Lys-Lys-Xaa-Lys (SEQ ID NO:59),Xaa-Lys-Lys-Xaa-Xaa-Lys-Lys (SEQ ID NO:60), Xaa-Lys-Lys-Xaa-Lys-Xaa-Lys(SEQ ID NO:61), Xaa-Lys-Lys-Lys-Xaa-Lys-Xaa (SEQ ID NO:62).Xaa-Lys-Lys-Xaa-Lys-Lys-Xaa (SEQ ID NO:63), Xaa-Lys-Xaa-Lys-Lys-Lys-Xaa(SEQ ID NO:64), Xaa-Lys-Lys-Lys-Xaa-Xaa-Lys (SEQ ID NO:65),Lys-Xaa-Lys-Lys-Lys-Xaa-Xaa (SEQ ID NO:66), Xaa-Lys-Lys-Lys-Lys-Xaa-Xaa(SEQ ID NO:67), Xaa-Lys-Lys-Lys-Xaa-Lys-Xaa (SEQ ID NO:68),Xaa-Lys-Lys-Lys-Xaa-Xaa-Lys (SEQ ID NO:69), Lys-Lys-Lys-Lys-Xaa-Xaa-Xaa(SEQ ID NO:70), Lys-Lys-Lys-Xaa-Xaa-Xaa-Lys (SEQ ID NO:71).Lys-Lys-Lys-Xaa-Lys-Xaa-Xaa (SEQ ID NO:72), Lys-Lys-Xaa-Lys-Lys-Xaa-Xaa(SEQ ID NO:73), Lys-Lys-Xaa-Xaa-Lys-Xaa-Lys (SEQ ID NO:74),Lys-Lys-Xaa-Xaa-Xaa-Lys-Lys (SEQ ID NO:75), Lys-Lys-Xaa-Lys-Lys-Xaa-Xaa(SEQ ID NO:76), Lys-Xaa-Lys-Lys-Xaa-Xaa-Lys (SEQ ID NO:77),Lys-Xaa-Lys-Xaa-Lys-Xaa-Lys (SEQ ID NO:78), Lys-Xaa-Lys-Xaa-Xaa-Lys-Lys(SEQ ID NO: 79), Lys-Xaa-Xaa-Lys-Lys-Xaa-Lys (SEQ ID NO:80).Lys-Xaa-Xaa-Lys-Xaa-Lys-Lys (SEQ ID NO:81), Lys-Xaa-Xaa-Xaa-Lys-Lys-Lys(SEQ ID NO:82), Lys-Lys-Xaa-Xaa-Xaa-Xaa-Lys,Lys-Xaa-Lys-Xaa-Xaa-Xaa-Lys, Lys-Xaa-Xaa-Lys-Xaa-Xaa-Lys,Lys-Xaa-Xaa-Xaa-Lys-Xaa-Lys, Lys-Xaa-Xaa-Xaa-Xaa-Lys-Lys,Xaa-Lys-Lys-Xaa-Xaa-Xaa-Lys, Xaa-Lys-Xaa-Lys-Xaa-Xaa-Lys,Xaa-Lys-Xaa-Xaa-Lys-Xaa-Lys, Xaa-Lys-Xaa-Xaa-Xaa-Lys-Lys,Xaa-Xaa-Lys-Lys-Xaa-Xaa-Lys, Xaa-Xaa-Lys-Xaa-Lys-Xaa-Lys,Xaa-Xaa-Lys-Xaa-Xaa-Lys-Lys, Xaa-Xaa-Xaa-Lys-Lys-Xaa-Lys,Xaa-Xaa-Xaa-Lys-Xaa-Lys-Lys, Xaa-Xaa-Xaa-Xaa-Lys-Lys-Lys,Lys-Xaa-Xaa-Xaa-Xaa-Xaa-Lys, Xaa-Lys-Xaa-Xaa-Xaa-Xaa-Lys,Xaa-Xaa-Lys-Xaa-Xaa-Xaa-Lys, Xaa-Xaa-Xaa-Lys-Xaa-Xaa-Lys,Xaa-Xaa-Xaa-Xaa-Lys-Xaa-Lys, Xaa-Xaa-Xaa-Xaa-Xaa-Lys-Lys,Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Lys, Lys-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa,Xaa-Xaa-Xaa-Xaa-Xaa-Lys-Xaa, Xaa-Lys-Xaa-Xaa-Xaa-Xaa-Xaa,Xaa-Xaa-Lys-Xaa-Xaa-Xaa, Xaa-Xaa-Xaa-Xaa-Lys-Xaa,Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa, wherein each Xaa is independently selectedfrom the group consisting of Ala, Leu, Ser, Thr, Tyr, Asn, Gin, Asp,Glu, Arg, His, Met, Orn, and amino acids of the formula I-NH—C(R¹)(R²)—C(═O)—  (I)  wherein R and R² are selected from the groupconsisting of hydrogen, C₁₋₆-alkyl, phenyl, and phenyl-methyl, whereinC₁₋₆-alkyl is optionally substituted with from one to three substituentsselected from halogen, hydroxy, amino, cyano, nitro, sulfono, andcarboxy, and phenyl and phenyl-methyl is optionally substituted withfrom one to three substituents selected from C₁₋₆-alkyl, C₂₋₆-alkenyl,halogen, hydroxy, amino, cyano, nitro, sulfono, and carboxy, or R¹ andR² together with the carbon atom to which they are bound form acyclopentyl, cyclohexyl, or cycloheptyl ring, e.g. 2,4-diaminobutanoicacid (Dbu) and 2,3-diaminopropanoic acid (Dpr).
 21. A peptide conjugateaccording to any one of the preceding claims wherein said amino acidresidues have an L-configuration.
 22. The peptide conjugate according toclaim 1, wherein said conjugate is selected from the group consisting ofdes Ser³⁹-exendin-4(1-39)-Lys₆-NH₂ (SEQ ID NO:91), desPro³⁶-exendin-4(1-39)-Lys₆-NH₂ (SEQ ID NO:93), desAla³⁵-exendin-4(1-39)-Lys₆-NH₂ (SEQ ID NO:94), desGly³⁴-exendin-4(1-39)-Lys₆-NH₂ (SEQ ID NO:95), desSer³⁹-(Lys⁴⁰(palmitoyl))exendin-4(1-39)-Lys₇-NH₂(SEQ ID NO:96), desGly³⁴-(Lys⁴⁰(palmitoyl))exendin-4(1-39)-Lys₇-NH₂ (SEQ ID NO:97), desAla³⁵-(Lys⁴⁰(palmitoyl))exendin-4(1-39)-Lys₇-NH₂ (SEQ ID NO:98), desPro³⁶-(Lys⁴⁰(palmitoyl))exendin-4 (1-39)-Lys₇-NH₂ (SEQ ID NO:99),Lys⁴⁰(palmitoyl)exendin-4(1-39)-Lys₇-NH₂ (SEQ ID NO: 100), desPro³⁶(Pro³⁷-exendin-4(1-39)Lys₆-NH₂(SEQ ID NO: 131), Lys₆-des Pro³⁶,Pro³⁷, Pro³⁸-exendin-4(1-39)-NH₂(SEQ ID NO: 134), Asn(Glu)₅-des Pro³⁶,Pro³⁷, Pro³⁸-exendin-4(1-39)-NH₂(SEQ ID NO: 137), Lys₆-des Pro³⁶, Pro³⁷,Pro³⁸-exendin-4(1-39)-Lys₆-NH₂(SEQ ID NO: 135), Asn(Glu)₅-des Pro³⁶,Pro³⁷, Pro³⁸-exendin-4(1-39)-Lys₆-N₂(SEQ ID NO: 136), des Pro³⁶,Pro³⁷Pro³⁸-exendin-4(1-39)-Lys₆-NH₂(SEQ ID NO: 133), Gly⁸-GLP-1(7-36)Lys₆-NH₂ (SEQ ID NO:88) Lys₆-Gly⁸-GLP-1 (7-36)Lys₆-NH₂(SEQ ID NO:118), Lys₆-Gly⁸-GLP-1(7-36)-NH₂(SEQ ID NO:119)(Gly⁸,Lys³⁷(palmitoyl)-GLP-1(7-36)(Human)-Lys₇-NH₂(SEQ ID NO:89),(Gly⁸,Lys²⁶(palmitoyl)-GLP-1(7-36)(Human)-Lys₆-NH₂ (SEQ ID NO: 103),Gly⁸,Lys³⁴(palmitoyl)-GLP-1 (7-36)(Human)-Lys₆-NH₂ (SEQ ID NO: 90),Gly⁸GLP-1 (7-36)-Lys₈-NH₂ (SEQ ID NO: 120), Gly⁸-GLP-1 (7-36)-Lys₁₀-NH₂(SEQ ID NO: 121), Gly⁸-GLP-1 (7-37)-Lys₆-NH₂ (SEQ ID NO: 122), and thefree acid thereof and a pharmaceutically acceptable salt thereof.
 23. Anovel peptide conjugate comprising X, a peptide agonist of GLP-1activity and/or exendin-4 activity, wherein X is selected from the groupconsisting of: des Pro³⁶-exendin-4(1-39)-NH₂ (SEQ ID NO:101), desPro³⁶-des Pro³⁷-exendin-4(1-39)-NH₂(SEQ ID NO: 130), des Pro³⁶-desPro³⁷-des Pro³⁸-exendin-4(1-39)-NH₂(SEQ ID NO: 132), desAla³⁵-exendin-4(1-39)-NH₂ (SEQ ID NO:105), des Gly³⁴-exendin-4(1-39)-NH₂(SEQ ID NO: 106), des Gly³⁴-(Lys⁴⁰ (palmitoyl))exendin-4(1-39)-NH₂ (SEQID NO:108), des Ala³⁵-(Lys⁴⁰ (palmitoyl))exendin-4(1-39)-NH₂ (SEQ IDNO:109), des Pro³⁶-(Lys⁴⁰ (palmitoyl))exendin-4(1-39)-NH₂ (SEQ IDNO:110), Gly⁸-Glp-1(7-36)-NH₂(SEQ ID NO: 87), and Gly⁸-Glp-1(7-36) (SEQID NO: 87), and wherein X is C-terminally bound via a peptide bond to apeptide sequence Z selected from the group consisting of (Lys)_(n) wheren is an integer from 4 to 8, preferably n is 6, and the free acidthereof and a pharmaceutically acceptable salt thereof.
 24. The peptideconjugate which is exendin-4(1-39)-Lys₆-NH₂ (SEQ ID NO:92) and the freeacid thereof and a pharmaceutically acceptable salt thereof.
 25. Apharmaceutically active conjugate of any one of claims 1 to 24 for usein therapy.
 26. A pharmaceutical composition comprising apharmaceutically active peptide conjugate as defined in any one ofclaims 1 to 24 and a pharmaceutical acceptable carrier.
 27. Apharmaceutical composition according to any one of claims 25 and 26wherein the active peptide conjugate is selected from the groupconsisting of des Pro³⁶-exendin-4(1-39)-Lys₆-NH₂ (SEQ ID NO:93),LYs₆-des Pro³⁶,Pro³⁷, Pro³⁸-exendin-4(1-39)-NH₂(SEQ ID NO: 134)Asn(Glu)₅-des Pro³⁶, Pro³⁷, Pro³⁸-exendin-4(1-39)NH₂SEQ ID NO: 137)Asn(Glu)₅-des Pro³⁶, Pro³⁷, Pro³⁸-exendin-4(1-39)-Lys₆-NH₂(SEQ ID NO:136), des Pro³⁶, Pro³⁷, Pro³⁸-exendin-4(1-39)-Lys₆-NH₂(SEQ ID NO: 133),and the free acid thereof and a pharmaceutically acceptable saltthereof.
 28. A pharmaceutical composition according to any one of claims25 and 26 wherein the active peptide conjugate is selected from thegroup consisting of Gly⁸-GLP-1 (7-36)-Lys₆-NH₂ (SEQ ID NO:88),Gly⁸-GLP-1 (7-36)-Lys₇-NH₂(SEQ ID NO: 117), and the free acid thereofand, a pharmaceutically acceptable salt thereof.
 29. A variant of aparent exendin, wherein said parent exendin has an amino acid sequencehaving at least an 90% homology to exendin-4 and wherein said variantlowers the blood glucose level and binds to a GLP-1 receptor in a mammaland has at least one modification selected from the group consisting of:(a) between one and five deletions at positions 34-38, and (b) containsa Lys at position 40 having a lipophilic substituent bound to theepsilon-amino group of Lys via an amide bond.
 30. The variant accordingto the preceding claim wherein said parent exendin is exendin-4.
 31. Thevariant according to the preceding claim wherein said exendin-4 isexendin-4(1-39).
 32. The variant according to any one of claims 29 to31, wherein said variant comprises a Lys at position 40 having apalmitoyl group bound to the epsilon-amino group of Lys via an amidebond.
 33. The variant according to any one of claims 29 to 32, whereinsaid variant is selected from the group consisting of desPro³⁶-exendin-4(1-39)-NH₂(SEQ ID NO: 101), des Pro³⁶ Pro³⁷Pro³⁸-exendin-4(1-39)-NH₂(SEQ ID NO: 132), des Pro³⁶Pro³⁷-exendin-4(1-39)-NH₂(SEQ ID NO: 130), desAla³⁵-exendin-4(1-39)-NH₂(SEQ ID NO: 105), desGly³⁴-exendin-4(1-39)-NH₂(SEQ ID NO: 106), des Ser³ ^(₉) -(Lys⁴⁰(palmitoyl) exendin-4(1-39)-NH₂(SEQ ID NO: 107), desGly³⁴-(Lys⁴⁰(palmitoyl)) exendin-4(1-39)-NH₂(SEQ ID NO: 108), desAla³⁵-(Lys⁴⁰ (palmitoyl) exendin-4(1-39)-NH₂ (SEQ ID NO: 109) and thefree acid thereof and a pharmaceutically acceptable salt thereof.
 34. Apharmaceutical composition comprising a variant of a parent exendin asdefined in any of the claims 29 to 33 and a physiologically acceptablecarrier.
 35. A pharmaceutical composition according to the precedingclaim wherein the variant is selected from the group consisting of desPro³⁶-exendin-4(1-39)-NH₂ (SEQ ID NO: 101) and the free acid thereof anda pharmaceutically acceptable salt thereof.
 36. The composition of thepreceding claim for use in therapy.
 37. A method for producing thepeptide conjugates of any one of claims 1-24 or the exendin variants ofany one of claims 29-33 having a natural polypeptide sequence,comprising a) introducing a nucleic acid sequence encoding a polypeptidesequence comprising the peptide sequence of said exendin variant or saidpeptide conjugate and a selectable marker contained within a nucleicacid construct or a vector into a host cell to obtain a recombinant hostcell; b) selecting said recombinant host cell; c) culturing saidrecombinant host cells under conditions permitting the production ofsaid polypeptide sequence; d) isolating said polypeptide sequence fromthe culture; and e) optionally cleaving said polypeptide sequence usingan appropriate protease to obtain said peptide conjugate.
 38. Apolypeptide sequence encoding the peptide conjugates of any one of thepreceding claims having a natural polypeptide sequence.
 39. Use of apeptide conjugate according to any one of claims 1-24 or an exendinvariant according to any one of claims 29-33 for the manufacture of apharmaceutical composition.
 40. Use of a pharmaceutically active peptideconjugate as defined in any one of claims 1-24 or an exendin variantaccording to any one of claims 29-33 for the manufacture of apharmaceutical composition for use in treatment of diabetes type I ortype 2, insulin resistance syndrome, obesity, eating disorders,hyperglycemia, metabolic disorders, and gastric disease.
 41. Use of apharmaceutically active peptide conjugate as defined in any one ofclaims 1-24 or an exendin variant as defined in any one of claims 29-33for the manufacture of a pharmaceutical composition for use in thetreatment of disease states associated with elevated blood glucoselevels elicited by hormones known to increase blood glucose levels, suchas catechol amines including adrenalin, glucocorticoids, growth hormoneand glucagon.
 42. Use of a pharmaceutically active peptide conjugate asdefined in any one of claims 1-24 or an exendin variant as defined inany one of claims 29-33 for the manufacture of a pharmaceuticalcomposition for use in regulation of blood glucose levels.
 43. Use of apharmaceutically active peptide conjugate as defined in any one ofclaims 1-24 or an exendin variant as defined in any one of claims 29-33for the manufacture of a pharmaceutical composition for use inregulation of gastric emptying.
 44. A method of stimulating insulinrelease in a mammal comprising administering an effective insulinotropicamount of the peptide conjugate of claims 1 to 24 or an exendin variantas defined in any one of claims 29-33.
 45. A method of lowering bloodglucose level in a mammal comprising administering an amount of thepeptide conjugate of claims 1 to 24 or an exendin variant as defined inany one of claims 29-33 effective to lower blood glucose level in saidmammal.
 46. A method of lowering plasma lipid level in a mammalcomprising administering an amount of the peptide conjugate of claims 1to 24 or an exendin variant as defined in any one of claims 29-33effective to lower plasma lipid level in said mammal.
 47. A method ofreducing mortality and morbidity after myocardial infarction in a mammalcomprising administering an amount of the variant of the peptideconjugate of claims 1 to 24 or an exendin variant as defined in any oneof claims 29-33 effective to reduce mortality and morbidity aftermyocardial infarction.
 48. A method of increasing the plasma half lifeof exendin 4 and exendin 4 variants comprising linking a polypeptidesequence of preferably 6 lysine residues via a peptide bond to theC-terminal of said exendin 4 or exendin 4 variant.